This study aims to determine the effects of Pleurotus ostreatus extract on Biofilms caused by Stenotrophomonas maltophilia and its significance on susceptibility and motility. Ethyl acetate method was performed for extraction procedure. Six (6) groups were assigned as positive group, negative group, and experimental group. Bacterial suspension was prepared on BHI medium. Experimental group were treated with different concentrations of the extract, Sulfamethoxazole (SXT) was used for the positive group and the negative group remained untreated. In the Biofilm assay, samples were dispensed on the wells of microtiter plates and was incubated then stained with crystal violet. In Motility assay, 1% of MHA was prepared, samples were dispensed on the surface of the 1% MHA for swimming motility. While the samples were pin inoculated at the bottom of the twitching plates. After 24 hours of incubation, growth was measured for swimming, and twitching plates were stained with Coomassie brilliant blue. For Susceptibility testing, the bacteria were inoculated on MHA, 10 ul of the extract and SXT were then dispensed onto the agar well. After 24 hours of incubation, zone of inhibition was measured. Results showed that Pleurotus ostreatus extract could not inhibit the biofilms, motility and multi-drug resistance. It was then concluded that Pleurotus ostreatus was considered as not effective in modulation of biofilms, motility and susceptibility of Stenotrophomonas maltophilia.