Tomáš Souček and Vladimír Coufal were on the wrong end of a 2-0 defeat for Czech Republic as they faced Portugal on Thursday night.
The West Ham United duo's country headed to Lisbon undefeated in UEFA Nations League Group A2, but they were unable to topple the league leaders.
Manchester City's João Cancelo opened the scoring 33 minutes into the contest, before a quickfire second from Valencia winger Gonçalo Guedes took the tie away from the visitors.
The three points keeps Portugal top of the table with seven points from their opening three games, while Czech Republic drop to third - although they will be encouraged by their start, which has seen them defeat Switzerland and draw with Spain.
Their campaign continues on Sunday when they remain on the Iberian Peninsula, heading to Malaga to face Spain.
