Also missing from the 23-man squad was Andre Gomes who, like Eder and Nani, played at the 2016 European championships won by the Portuguese.
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Eder and Nani miss out on Portugal World Cup squad
Forwards Eder and Nani were surprise absentees from Portugal's World Cup squad with national team coach Fernando Santos opting instead for the youth of Andre Silva and Goncalo Guedes to play alongside superstar Cristiano Ronaldo.
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Injured Porto midfielder Danilo Pereira was also missing from the line-up.
At 33, five-time Ballon d'Or winner Ronaldo and 34-year-old Ricardo Quaresma are the only players over 30 in the Portuguese attack.
Eder scored the extra-time winning goal in the Euro 2016 final against France but he was desperately out of form for Lokomotiv Moscow this season, scoring just four goals as his club side claimed the Russian Premier League title.
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Nani, 31 and his country's third most-capped player behind Ronaldo and Luis Figo with 112 appearances, has played just 24 matches all season with Lazio.
"From a human perspective, it's very hard to discard players who were with us at Euro 2016," admitted 63-year-old coach Santos.
"They have all contributed to writing a brilliant chapter in the history of Portuguese football. But I had to make choices that for me make a better puzzle."
Winger Guedes, 21, has impressed on loan at Valencia from Paris Saint-Germain this season, having struggled in France last term.
Andre Silva, 22, scored eight goals in 14 games during AC Milan's Europa League campaign, which proved enough to see him called up despite only starting seven Serie A matches.
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Santos called up 21-year-old Benfica defender Ruben Dias for the first time, but Monaco youngster Rony Lopes missed out despite a breakthrough season in Ligue 1 with 13 goals from midfield.
Wolverhampton Wanderers midfielder Ruben Neves also failed to make the final list, despite starting a friendly win in March over Egypt.
Portugal are in Group B at the World Cup, which starts in Russia on June 14, along with former champions Spain, Morocco and Iran.
Before the finals, they will play friendlies against Tunisia, Belgium and Algeria.
Portugal squad:
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Goalkeepers: Anthony Lopes (Lyon/FRA), Beto (Goztepe/TUR), Rui Patricio (Sporting Lisbon)
Defenders: Bruno Alves (Glasgow Rangers/SCO), Cedric Soares (Southampton/ENG), José Fonte (Dalian Yifang/CHN), Mario Rui (Napoli/ITA), Pepe (Besiktas/TUR), Raphael Guerreiro (Borussia Dortmund/GER), Ricardo Pereira (FC Porto), Ruben Dias (Benfica)
Midfielders: Adrien Silva (Leicester City/ENG), Bruno Fernandes (Sporting Lisbon), Joao Mario (West Ham/ENG), Joao Moutinho (Monaco/FRA), Manuel Fernandes (Lokomotiv Moscow/RUS), William Carvalho (Sporting Lisbon), Bernardo Silva (Manchester City/ENG)
Attackers: Andre Silva (AC Milan/ITA), Cristiano Ronaldo (Real Madrid/ESP), Gelson Martins (Sporting Lisbon), Goncalo Guedes (Valencia/ESP - on loan from Paris Saint-Germain/FRA), Ricardo Quaresma (Besiktas/TUR)
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