Ronaldo was left emotional at the final whistle after Mikel Merino scored a late winner to send Spain into the quarter-finals.
The 41-year-old Portuguese star is speculated to be calling an end to his international career after this World Cup.
A solitary goal proved enough to separate the two sides in a tightly contested encounter, with Spain producing a disciplined defensive display to protect their lead.
Portugal pushed hard for an equaliser in the closing stages but failed to breach Spain's organised backline.
The victory sets up a quarter-final clash between Spain and Belgium, while Portugal's World Cup campaign comes to an end.
Belgium thrash USA to book quarter-final berth
In the other Round of 16 fixture, Belgium national football team stormed into the FIFA World Cup quarter-finals with an emphatic 4-1 victory over the United States men's national soccer team in Seattle.
Charles De Ketelaere starred with two goals and an assist as Belgium capitalised on a series of costly defensive errors by the hosts.
Malik Tillman briefly restored parity for the USA with a well-taken free-kick after Belgium's early opener, but De Ketelaere quickly put the Europeans back in front before Hans Vanaken extended the lead in the second half.
Romelu Lukaku added a stoppage-time goal to seal the comprehensive victory, setting up a mouth-watering quarter-final showdown against Spain while bringing the United States' World Cup campaign to an end.