Referee Alejandro Hernández Hernández walked over to Madrid's technical area to caution the head coach after Mourinho reacted angrily to a contentious call moments into the second period, in a match that had already been simmering with discontent from the away side.
Vinícius Júnior had protested several decisions in the opening exchanges after the break, and Arda Güler was shown a yellow card of his own, compounding a growing sense among the Madrid players that the officiating was working against them.
Mourinho's booking came as that frustration reached its peak, with the Portuguese remonstrating loudly from the touchline before the referee intervened directly.
The moment underlined the tension running through a game Real Madrid had dominated for long spells but ultimately lost 1-0, their first defeat since Mourinho's return to the club. It capped an ill-tempered afternoon in which five Madrid players were shown yellow cards in total, a tally Mourinho later suggested reflected contrasting approaches to gamesmanship between the two sides rather than any players stepping notably out of line.
The flashpoint added to a wider narrative of frustration around decision-making in the match, including a VAR-disallowed goal for an offside in the build-up and a stoppage-time penalty save that went unreviewed for encroachment.
Real Madrid's players left La Cartuja with a clear sense of grievance over the officiating, even as Mourinho himself was careful afterwards not to place the blame for the result squarely on the referee.
It is the kind of touchline flashpoint that has followed Mourinho throughout his career, with confrontations with match officials a recurring feature of his time at clubs including Chelsea, Manchester United and Inter Milan. His return to Real Madrid this summer had so far been notably free of such incidents, with three wins from three league games played in relatively straightforward circumstances before Friday's contentious atmosphere at La Cartuja.
The booking means Mourinho will need to be mindful of his conduct in the technical area heading into Madrid's next fixture, with any further disciplinary issue potentially triggering closer scrutiny from LaLiga's disciplinary committee.