Betfair will cease operations in the country and will no longer allow new players to register. William Hill and Amaya-owned brands Full Tilt and PokerStars have already confirmed their exit from the national market.
The series of withdrawals comes after Portugal in June began operating under a new online gambling regime.
Companies that wish to continue operating in Portugal are now in the process of withdrawing their services from the country ahead of a new licence application process that is due to begin in September.