Objective: Free-living amoeba (FLA) are protozoa living in soil and in natural and man-made water systems. They attract much attention owing to the illnesses associated with them and to their relationships with bacteria. In this study, the effect of cell-free supernatant (CFS) obtained from FLA against Staphylococcus was investigated.Materials and Methods: Environmental FLA strains (A1, A2, A3) were obtained from lake water and swimming pools in Istanbul. Acanthamoeba castellanii ATCC 50373 was used as the standard strain. Clinical Staphylococcus strains (S1, S2, S3) were obtained from a culture collection at Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology. As standard strains, MRSA ATCC 43300, S. epidermidis ATCC 12228, S. aureus ATCC 29213 were used. FLA-CFS were obtained by centrifuging and filtering of axenic cultures. Colony counting technique was used to investigate the inhibition activities of FLA-CFS against Staphylococcus bacteria.Results: Against MRSA ATCC 43300 strain, CFSs of A. castellanii ATCCC 50373 and A1 showed an inhibition efficiency of 78.36% and 73.47%, respectively. Against S1 strain, CFSs of A. castellanii ATCCC 50373 and A2 showed an inhibition of 65.64% and 15.14%, respectively. Against S. aureus ATCC 29213, only A1-CFS showed inhibitory effect (44%). It was found that A. castellanii ATCC 50373 and A2-CFSs inhibited the S2 strain 26.20% and 9.24% respectively. Against S3 strain, A2-CFS was inhibitory at 33.33%. No FLA-CFS could be inhibitory against S. epidermidis ATCC 12228. Conclusion:It is necessary to devise new studies in which sample numbers are increased when using FLA strains in the inhibition of antibiotic-resistant bacteria.