The hosts lost to fourth-tier Valadares Gaia after extra-time, while the Gilistas progressed against Serpa from the same level with relative ease.
Match preview
Chaves had beaten Braga in their last league game, which ended a four-game run without a win thanks to Hector Hernandez's second-minute strike.
They will have lost much of the momentum they built from that game with the 3-2 defeat at Valadares Gaia, in a game that saw Ricardo Guimaraes equalise in the 94th after the hosts had taken the lead just two minutes before.
The Flavienses had Helder Morim sent off in extra time before conceding eight minutes from the end from the penalty spot.
Their league form has been satisfactory this term following their promotion from the second tier in May, with an even number of wins, draws and defeats and victories against Braga and Sporting Lisbon to their name.
They are yet to win at home, though, with their last home win coming in the first leg of their playoff victory over Moreirense in May, and a win against Gil Vicente could take them 10 points clear of the drop zone.
This weekend's visitors used the break in league action to their benefit last weekend, picking up their first win in four games at the fourth-tier side.
They currently occupy 14th in the table with just two wins to their name, both of which came against the current bottom two in Pacos Ferreira and Maritimo.
Five of their eight goals scored have come away from home, and the win against basement side Maritimo also came on the road.
They have lost two of their four games away from home this season, conceding seven of their 12 goals on their travels.
Vicente started the season in the Europa Conference League before being resoundingly eliminated by AZ Alkmaar in August, but are now a long way off from qualifying for Europe once again and are starting to look over their shoulders at the teams below.
Chaves Primeira Liga form:
W
D
L
L
D
W
Gil Vicente Primeira Liga form:
D
L
W
D
L
L
Team News
After his red card in the cup last time out, Morim will miss this game through suspension.
Chaves' left-back Bruno Langa is a serious doubt for the game, which could see Sandro Cruz start in his place in a straight swap, but other than that they have a full squad to choose from.
Ali Alipour's goal and assist in the cup could see him force his way into contention, but Ivo Vieira will likely keep Fran Navarro as his leading frontman.
Murillo was rested for the cup game so should be able to rejoin Navarro and Juan Manuel Boselli in the front three.
Chaves possible starting lineup:
Vitor; Correia, Monte, Vitoria, Cruz; Mendes, Pina, Teixeria; Juninho, Hernandez, Arriba
Gil Vicente possible starting lineup:
Andrew; Veiga, Cunha, Fernandes, Marin; Fujimoto, Carvalho, Tiba; Boselli, Navarro, Murillo