In short, yes, they did have trouble reading the manuscripts, depending upon the specific script, the age of the manuscript, and how heavily abbreviated it was, amongst other issues. Since you are asking about the Renaissance, two of the best sources for quotes about the issue would be the famous Humanists Petrarch and Poggio Bracciolini. Petrarch, famously the leader of the Florentine Humanists, complained about the prevailing script of his day:

oculos ... fatigans	it fatigues the eyes	Petrarch, Epistolae familiares. XXIII.18.8
perexiguis atque compressis visumque frustrantiubus literulis ... acervans omnia et coartans tantium agges- tione confundens.	the letters are small and compressed and frustrating to look at ... packed-in and piling-up like so much pouring sand	Petrarch, Seniles, VI.5.

This prevailing script was presumably Gothic or Blackletter, a large family of similar scripts largely characterised by angular, upright, highly-compressed writing, where it could be hard to tell many characters apart, since a large number of characters were made of "minims", a single stroke that is the height of the lower-case letter, with small angular strokes on the top or bottom. It can be difficult if not impossible to tell whether a character is a u or an n without knowing the word surrounding it, similarly, vi, m, ni, ui, iv, in, iu all look very similar. Appropriately, the word minimum is all minims. Try to read this: ııııııııııııııı. In practice, there is generally a small amount of space or linking that distinguishes letters, but that worst-case scenario gives you an idea of the kind of problems that one might face.

But, there's more! In addition to the wide variety of Gothic hands of varying legibility, there were also a great number of cursive hands and specifically hard-to-read variants known as 'chancellery' or 'secretary' hands, used for recording legal acts, records, and accounts, produced by and for professional scribes with potentially large workloads who wrote for their own legibility, not for general legibility, as is the case with what we call a 'book' hand. Even worse, there are old and disused scripts and practices that would likely not be familiar to contemporary readers, and required deciphering. Poggio in his Letter LXXXII to Niccolo Niccoli refers to a manuscript of Plautus' plays so:

The book is in an ancient and corrupt hand ... and there are many imperfections in it. I shall not have it copied before I have read it and corrected it for, unless it is copied by a learned man, all the work will be in vain.

Since all books were manuscripts, the quality and education of the copyist was very much a concern. If a copyist had a bad hand or was a poor reader, there are all kinds of errors that they might make, on top of the normal range of scribal errors (homeoteleuton, homeoarchy, haplography, dittography, etc.). Poggio writes in Letter VIII to Franciscus Barbarus:

The man who copied the books was the most ignorant of living men; one needs to use divination, not reading itself, and so it is very important that they be copied by a scholar.

If a Renaissance reader came across a truly ancient text (not a mediated copy), there might be other problems: many Ancient texts have no word separation, and relied upon either familiarity with the text or familiarity with the poetic metre to ease reading. Books did not have to be ancient, however, to be problematic: many scripts proliferated in the past and had disappeared by the time of the Humanists. A notable one is Beneventan, which Poggio refers to as "Lombard", a script that was used solely, so far as I know, at Monte Cassino and its daughter houses, around the High Middle Ages. It is defunct as a script by the time of Poggio and is generally harder to read, though not so difficult as, say, Visigothic or New Roman Cursive.

In his Letter LII to Niccolo Niccoli, Poggio says

You have sent me the Seneca and the Cornelius Tacitus which I am glad to have. But the latter is in the Lombard script and for the most part illegible; if I had known that I would not have given you the trouble. Once when I was staying with you I read another copy written in ancient script; I do not know whether it belonged to Coluccius or to somebody else. I should like to have that or some other book which can be read, for it will be difficult to find a copyist who can read this volume correctly; so try to let me have another one if it can be done.

Note that what Poggio refers to as 'ancient script' and what he uses as the model for the creation of Humanist Minuscule, which in turn becomes our Roman typeface, predecessor of what you are reading now on your screen, was not actually ancient, but rather Carolingian Minuscule, which the Humanists found aesthetically pleasing and easy to read and mistook for ancient script. Also, Poggio seems to be able to read Beneventan fine in other letters (compare Letter LIV, where he recommends volumes in a Lombard hand to be pursued by his fellows) so the concern here may not be for himself, but rather for the scribes that he seems to have employed in copying newly-rediscovered ancient texts and disseminating them to his fellow Italian Humanists.

I have not even gotten around to the difficulties with heavily-abbreviated texts, but this should suffice to make it clear that there were many reasons why Renaissance readers might find it difficult to read manuscript texts. These issues drove the development of humanistic minuscule, which, I have noted above, we eventually adopted for print (though many early printed works were in Gothic, and German presses used it up through WW2).

Poggio letters numbering and translations according to: Bracciolini, Poggio. Two Renaissance Book Hunters: the Letters of Poggius Bracciolini to Nicolaus De Niccolis ; Translated From the Latin and Annotated by Phyllis Walter Goodhart Gordon. New York : Columbia University Press, 1991. https://hdl.handle.net/2027/heb.05978