Nacional Asuncion had not lost in five games in Paraguay's Division Profesional but failed to continue that streak against Libertad, with Blas Diaz scoring the winner for the visitors just after the hour-mark.
Libertad rose to 32 points with their win at Nacional's Estadio Arsenio Erico, while the hosts remain on 29, dropping to fourth on the table.
A third straight win sees Libertad, who have a game in hand, just two points behind second-placed Deportivo Capiata (34 points).
Cerro Porteno remain top of the Division Profesional standings on 44 points after they came from behind to win 2-1 over Sportivo Luqueno.
Derlis Alegre gave Luqueno the lead after 24 minutes before Fidencio Oviedo equalised for Cerro Porteno in the 53rd minute.
Just eight minutes later and the league leaders were in front thanks to a goal from Jose Maria Ortigoza, which extended Cerro Porteno's undefeated run to 16 matches.
Capiata had the biggest win on Wednesday with a brace from Julio Santa Cruz setting up their 3-1 triumph over Cerro Porteno PF, while Sol de America drew 1-1 with General Diaz and Olimpia overcame Rubio Nu 1-0.
On Tuesday, Guarani played out a scoreless draw against Deportivo Carapegua.
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