Resistance-Nodulation-Division (RND) efflux pumps are relevant determinants ofStenotrophomonas maltophiliamultidrug resistance as they can extrude a broad range of antibiotics and compounds involved in virulence and physiological functions.S. maltophilia, an environmental bacterium, was shown to be associated with amoebae and able to multiply inside them. To explore whetherS. maltophiliaRND efflux pumps play a role when interacting with amoebae, we evaluated the effect of amoebal culture and co-culture supernatants on the growth ofS. maltophiliaand the expression ofsmeefflux pump genes.Acanthamoeba castellaniiandWillaertia magnawere used as amoebal models and strainS. maltophiliaBurE1 as bacterial one. Our data showed that both bacterial growth andsmegene expression were not modified by amoebal culture supernatants. On the contrary, co-culture supernatants negatively impacted the growth of BurE1 and induced the expression of three out of eight efflux pump genes, i.e.smeE,smeN andsmeZ. Finally, we evidenced the production ofA. castellaniisecondary metabolites, putatively belonging to the diterpene family, in the amoebal supernatant and in the co-culture supernatant ofA. castellaniiand BurE1. Whether these compounds act directly as substrates of the efflux pumps and/or inducers of thesmegenes need further investigations.