In The Road to El Dorado, a guard tries to arrest the protagonists for playing with loaded dice. Was it actually illegal to cheat on games like this in 15th century Spain? What kind of trial, if any, would have awaited Miguel and Tulio had they been captured?

Been waiting a few days myself for a response, so I'll share the sources I've found so far. To the mods: I am not a professional historian, and Spanish history and gambling culture is not my forte.

Gambling in Spain was certainly extremely widespread. In 1276, the Ordenamiento de las Tafurerías under Alfonso X was explicitly designed to fulfill the need for a law to regulate gambling and promote social harmony (as gambling and associated behaviors could quite obviously cause significant harm).

This law explicitly enumerates penalties for a variety of behaviors, being especially concerned with blaspheme by those engaged in gambling. Cards and dice were also regulated by the law; used weighted or otherwise altered dice results in a payment of a double indemnity as well as court costs. If you are unable to pay the double indemnity, the punishment was 30 lashes for the first offense and fifty lashes for the second offense. Repeatedly using altered dice would be punished by wearing the dice around your neck, being flogged through the streets, and finally, banishment.

However, here it should be noted that the laws were intended to apply to Royal gambling houses, not private ones. Alfonso X may have sought to monopolize the revenue from gambling by providing regulated, taxed houses with clear regulations, as Law 26 forbids unregulated establishments and frequenting gaming houses not under the Royal charter. Going to an illegal gambling parlor (a street dice game would likely have qualified) would have carried a fine of 10 maravedis. Informants about illegal gambling received one-third of all fines levied against the illegal games, and if they were a participant, gained immunity in exchange for testimony. The alcades who adjudicated against illegal gambling would also receive 1/3 of the fines imposed. Those who can't pay the fines would receive 200 lashes, with the whip dipped in water after each lash. Yes, they really didn't want you gambling without paying taxes to the Crown.

The above relates to the 1276 law; John I updated gambling laws in 1387, but I was unable to find the full text. A source below claims it banned all gambling; but other sources contradict this, stating that Jesuit commentators were astonished by the number of cards houses selling cards in Madrid, and that the state still received revenues from gambling, and that the law only restricted some types of gaming. However, it appears that gambling's appeal did not change, as the Spanish crown continued to receive a healthy revenue from taxing gaming, and regulating the sale of cards, dice, and other tools. Spanish fiction throughout the period frequently depicts cards, gaming, and gambling, with many of the familiar stereotypes of the "loser" gambler and conflicts and social ills it can bring, and there was always a strong desire to regulate the industry, while recognizing it could not be stopped entirely, despite constant moralizing and agonizing over blaspheme and secular concerns.

A few other sources that popped up mention the frequent discoveries of dice in archaeological digs, dice without 1s or 2s in Norway, dice loaded with Mercury on display in London, and other loaded dice almost certainly used for cheating. These dice and prohibitions against them have likely been around since dice and games of chance have existed. Punishments were severe, and in the case of private dice games not adjudicated by the Crown, the violence accompanying such a dice game is a frequent staple of commentaries and depictions of the day.

TL;DR Miguel and Tulio likely would have been punished for gambling outside of an officially chartered Royal gaming house, as such games were illegal. Cheating in dice was illegal, and punished. If you cheated in an Royal gambling house, you would have a trial. I cannot speak to the fairness of the trials, or how it would differ for an offense at a chartered gambling house vs. a non-charted one, or if cheating at an unofficial house would be fined in addition to the fine of the illegal game. Hopefully an expert on medieval Spanish law could chime in there. Whether or not the 1387 law banned all gambling or not, there is no scenario in which their game of street dice was legal, or in which using loaded dice was legal. I cannot comment on how the laws would interact, or affect the nature of their trial.

Sources: Fickle Fortune: Gambling in Medieval Spain, https://www.jstor.org/stable/4174306

Outside Bets: Disciplining Gamblers in Early Modern Spain https://www.jstor.org/stable/40541418

Dice https://medievallondon.ace.fordham.edu/collections/show/92#_ftn9

Gambling in Spain: Update on experience, research, and policy http://diposit.ub.edu/dspace/bitstream/2445/65784/1/630977.pdf