The defending champions were narrowly undone by Belgium in Seville.
Thorgan Hazard scored the winner shortly before half-time and, despite Portugal’s best efforts, they were unable to find an equaliser.
Ronaldo had scored five times in his three group games but could not add to that tally in the last 16.
The 36-year-old saw a powerful first-half free-kick saved by Thibaut Courtois but otherwise failed to bother the Belgian goalkeeper.
Portugal spurned several opportunities with Raphael Guerreiro coming closest as his strike cannoned off the post late on.
Ronaldo’s frustration was evident at the full-time whistle as he threw his captain’s armband down before kicking it.
Another individual had to pick it up off the ground as the Juventus star trudged down the tunnel.
Portugal boss Fernando Santos said: “There are players crying in the changing room. They gave everything. Belgium had 6 shots, we had 29. Sincerely I don’t have many words.
“We believed if we won this one, we would have gone on to win the next one. They scored, we didn’t. It’s football.”
“The players gave everything. They were tired but found energy. I picked Palhinha to try and freshen up the team. The same with Dalot over Semedo, who has been very good in training.”
Ronaldo had matched the international goalscoring record held by Iranian Ali Deai following his efforts at the Euros, but that is likely to be of little solace.
Attention will now turn to where the forward will play his football next season with his future at Juventus far from certain.
Belgium defender Thomas Vermaelen, formerly of Arsenal, conceded that his side were perhaps fortunate to get over the line as they withstood second-half pressure.
“In the first half we were playing high and putting pressure on Portugal, but looking at that second half, we are lucky to win,” he said.
“Portugal made it really hard for us. We had to really stay focused. It has cost us a lot of energy, but we have some time to recover and now we are looking forward to the game against Italy.”
The Italians have gone unbeaten in their last 31 games but needed extra-time to beat Austria at Wembley on Saturday night.
They and Belgium are in the tougher side of the draw and whoever progresses are likely to face France or Spain in the last four.