Substitute Mikel Merino settled a tight last-16 tie in the first minute of stoppage time, latching onto a Ferran Torres pass to beat Diogo Costa at his near post and send Spain into the quarter-finals.
It proved the final chapter of Ronaldo's remarkable World Cup story, one that stretched back two decades to his debut goal against IR Iran at Germany 2006.
"I'm fine, sad to leave the World Cup this way. But, as I said yesterday in the press conference, I gave my best and I leave with a clear conscience," Ronaldo told SportTV.
"That's the life of a football player. Sometimes we win, sometimes we lose and we need to keep going. The truth is that it was my last World Cup, and now I will have time with my family so as not to make decisions in the heat of the moment."
"I will get up tomorrow with a clear conscience. I won three titles for Portugal - one European Championship and two UEFA Nations Leagues. Before Cristiano, Portugal had never won any titles. Therefore, I leave with a clear conscience. I gave my best. Tomorrow will be a new day, and life goes on."
The 41-year-old, now playing his club football in Saudi Arabia, went on to feature - and score - at every finals since his 2006 bow, including South Africa 2010, Brazil 2014, Russia 2018 and Qatar 2022.
He made history in the group stage of this tournament, netting against Uzbekistan to become the first player to score at six different World Cups.
Ronaldo's 27 appearances at the finals leave him second on the all-time list, with 11 goals to his name.
Moments after the final whistle, Martinez confirmed he too was leaving his post, with his contract expiring on the day of Portugal's elimination.
"I came to Portugal to win the World Cup and I think that, without winning it, there's no point in continuing," Martinez said. "The board and the president now have the opportunity to choose the new manager. My contract ends today. There isn't much more to say."
The Spaniard, who took charge in 2023, also led Portugal to the quarter-finals of UEFA EURO 2024 and glory in the UEFA Nations League last year.
"Yes, it's my last game for the national team," he added. "I've felt welcomed as just another Portuguese person, in a very warm way. It's been a pleasure, a source of pride and a responsibility."