Jayne Ludlow's team beat the hosts 2-1 on Thursday, but fell victim to the game's only goal on Sunday.
Portugal replacement Claudio Neto struck in the 68th minute to break the deadlock.
Wales went on tour to prepare for the Women's World Cup qualifying campaign, which begins in the autumn.
England, Russia, Bosnia & Herzegovina and Kazakhstan will be Wales' group one opponents.
Wales' final warm-up game, against Netherlands, will take place on Saturday, 8 July.
Ludlow's substitutions in the opening game included a senior debut for Wichita FC forward Peyton Vincze.
Team: Laura O'Sullivan, Rhiannon Roberts, Sophie Ingle (capt), Gemma Evans, Hayley Ladd, Bronwen Thomas, Kayleigh Green, Angharad James, Loren Dykes, Natasha Harding, Chloe Chivers.
Subs: Hannah Miles, Charlie Estcourt, Nadia Lawrence, Claire Skinner, Shaunna Jenkins, Georgia Evans, Emma Beynon, Melissa Fletcher, Emma Gibbon, Peyton Vincze, Ffion Morgan.
Wales Women ended their two-match friendly series in Portugal with a 1-0 defeat at Stadium Municipal in Mangualde.