The Portuguese Football Federation announced on Thursday that the forward has been ruled out of the World Cup warm-up game in Foxborough, Massachusetts, with tendinitis and a muscle injury in his left leg.
Raul Meireles and Pepe will also miss their country's penultimate friendly before the team heads to Brazil.
The Seleccao face the Republic of Ireland in New Jersey in their final warm-up game, before taking on Germany in their Group G opener on June 16.
Ronaldo led Madrid to the Champions League title last month despite being troubled by leg injuries late in the season.
Portugal will also face the United States and Ghana in a tough looking group in Brazil.
The match with Portugal, meanwhile, is Mexico's final friendly before facing Cameroon in Group A on June 13.
El Tri will hope to bounce back from a poor showing in their 1-0 defeat against Bosnia on Tuesday.