The national team's players, coach Luis Enrique and staff have been forced to train individually this week at the team's training headquarters outside Madrid since captain Busquets was sent home to self-isolate on Sunday. Shortly after the match, the Spanish federation confirmed Diego Llorente had also tested positive.
The friendly against Lithuania -- Spain's final warm-up match ahead of Euro 2020 -- went ahead as a full international, but with members of Spain's under-21 squad taking part, coached by their manager Luis de la Fuente.
The Under-21s, who were knocked out of their own European Championships by Portugal at the semifinal stage last week, had to be called back from their holidays to take part.
Only winger Bryan Gil had previously featured for the senior side, making his debut in March, with the other ten starters and six substitutes all earning their first caps.
"I'm really happy. I've fulfilled a dream, just like the players," De La Fuente said in his post-match news conference. "I enjoyed it. I feel proud to have coached the senior team once... I want to thank the federation for the opportunity and I hope I can repeat it in the future!"
Valencia's Hugo Guillamon and Real Madrid's Brahim Diaz scored in the first half at Leganes' Butarque stadium, and Braga's Abel Ruiz missed a penalty, before substitutes Juan Miranda and Javi Puado grabbed a third and fourth after half time.
The Spain squad are being tested daily ahead of Spain's Euro 2020 opener with Sweden in Seville on Monday, with all players and staff returning negative results so far.
Six senior players -- Kepa Arrizabalaga, Raul Albiol, Brais Mendez, Pablo Fornals, Rodrigo Moreno and Carlos Soler -- were called up to train in a parallel bubble should multiple replacements be required.