Congress MLAs Pravin Maru, Pradyumansinh Jadeja, Soma Koli Patel, JV Kakadia and Mangal Gavit have submitted their resignations to the Gujarat Assembly Speaker Rajendra Trivedi.
Earlier, the Congress had suspended these five MLAs from the primary membership of the party after they resigned as legislators from the Assembly.
The resignations came days ahead of biennial Rajya Sabha elections in the state.
In the 182-member Gujarat Assembly, the BJP has 103 seats while the Congress has 68, after the resignations of five of its MLAs.
The BJP has fielded three candidates, including former Deputy Chief Minister Narhari Amin, as its candidate for the Rajya Sabha election.
Voting and counting for the biennial Rajya Sabha elections are scheduled to be held on March 26.