The current study was aimed to study the effects of both synergists S.S.S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) on permethrin tolerance in Anopheles gambiae s.l. larvae from Athiémé district in mono department in south-western Benin, West Africa. Larvae and pupae were collected from March to July and August to November 2018 during the rainy season in Athiémé district. Larval bioassays were performed on these collected Anopheles gambiae s.l. larvae using permethrin as larvicide and S.S.S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) as enzymes inhibitors or synergists. The results showed that both esterase and mono-oxygenase enzymes played a role in Anopheles gambiae s.l. larvae tolerance to permethrin in Athiémé district.