The 41-year-old failed to add to his men's-record international haul of 143 goals, despite being presented with clear-cut chances with just the goalkeeper to beat in both halves in Leiria on Wednesday (Thursday AEST).
The five-time world player of the year was substituted off in the 65th minute, having been given longer on the field than every other outfield player to start the match for Portugal.
Ronaldo will be making his record-tying sixth appearance at a World Cup.
Francisco Conceicao, one of 11 replacements to come on for Portugal, grabbed the 75th-minute winner by cutting in from the right and curling a shot into the far corner.
Pedro Neto gave Portugal the lead in the 23rd minute. Akor Adams equalised 14 minutes later for Nigeria, who failed to qualify for the World Cup.
Portugal are in Group K at the World Cup with Congo, Uzbekistan and Colombia.
In Florida, the returning Declan Rice set England on course for a morale-boosting 3-0 victory against Costa Rica after a heavy thunderstorm in Orlando delayed their final World Cup warm-up match by an hour.
Having arrived in Florida at the start of last week and scraped a 1-0 win in Saturday's workout against New Zealand, Thomas Tuchel's side stepped up preparations a week out from their curtain-raiser against Croatia.
Kick-off was put back an hour after a storm battered Orlando in the build-up to Wednesday's friendly, and England wasted no time when play finally got under way as vice-captain Rice gave them an early lead.
Anthony Gordon set up the returning Arsenal star to score in the ninth minute, before the Barcelona new boy slammed home a second-half penalty as Tuchel's men finally made their dominance count in a one-sided - and at times testy - encounter.
Ollie Watkins added another as the England boss got an encouraging response from his substitutes, having changed all 11 players again, albeit after the hour-mark, having done so at halftime in Tampa.
Meanwhile, Algeria forward Amine Gouiri struck twice in their 4-0 win over Bolivia in their World Cup warm-up in Kansas City.
- with PA, Reuters
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