During the Renaissance, the soldiers of the newly unified Spain fought with the Moors for control of Granada, and enforced the Spanish Inquisition. Ezio Auditore da Firenze came into conflict with many guards when he came to the country to rescue several Spanish Assassins, who had been arrested by the Inquisition after Rodrigo Borgia had given Tomas de Torquemada a list of their names.
In 1507, Spanish forces were engaged in a civil war, where Louis de Beaumont defended Viana from an attack by Cesare Borgia, who commanded a mainly-Spanish army on behalf of King John of Navarre. Ezio came to assassinate Cesare, but was attacked by troops on both sides who mistook him for an enemy. Despite this, Ezio assassinated Cesare, earning him Ferdinand's gratitude.
During the Age of Discovery, Spanish soldiers and scholars known as conquistadors conquered the New World. Among them, the Assassin Giovanni Borgia served Hernán Cortés under an assumed name during the conquest of Mexico. Whereas the conquistadors mostly used this as an opportunity to loot gold from the Aztecs, Giovanni obtained a Crystal Skull.
Around the Golden Age of Piracy onward, the Spanish Army was widely used in the Templars' activities in the Caribbean, mainly because the Grand Master of the Caribbean Templars was a Spanish governor. The Templars sent both Spanish and British soldiers to frequently attack the Assassin hideout of Tulum. They were also assigned to guard Templars and accompanied Laureano de Torres y Ayala when he visited the Observatory.
However, the Spanish were unpopular with the Louisianan citizens, causing a rebellion when they were blamed for people disappearing. The Assassin Aveline de Grandpré antagonized them to draw the attention of governor Antonio de Ulloa, from whom she learned that the disappearances were a Templar plot to divert slaves for Rafael Joaquín de Ferrer, in order to excavate ruins in Chichen Itza.
Some Spanish soldiers were later bribed by Diego Vázquez to occupy the Louisiana Bayou in an effort to eliminate the Assassin Mentor Agaté, and to seize control of smuggling operations in the region. Later, he used his rogue soldiers to attempt to block Spanish supplies for the Patriots in British America, but this did not deter governor Bernardo de Gálvez from siding with the Continental Army during the American Revolutionary War.
- In several trailers in Assassin's Creed IV: Black Flag, Spanish soldiers had a much more different appearance. They wear white uniforms and had a white bow in their collars which would have shared the appearance the early versions of British Redcoats but their appearance was changed to Yellow due to historical inaccuracies since the French Army's main colors were white. These 'Whitecoats' appeared as officials that could be bribed in the final game. These White uniformed soldiers later appeared as the French Army at Freedom Cry.