Recently, the search for novel products derived from natural sources has become necessary due to the decreasing effectiveness of current antibiotics in treating bacteria that are antibiotic-resistant. In this context, it is well known that entomopathogenic bacteria (EPBs) produce a broad range of secondary metabolites with antibacterial activities. Therefore, an in-vitro trial was conducted to isolate and identify non-symbiotic bacteria associated with entomopathogenic nematodes, Steinernema spp. and evaluate the antibacterial activity against four antibiotic-resistant bacteria. Four bacterial isolates, i.e. Pseudomonas alcaligenes ST-1, Paenibacillus barcinonensis ST-2, Bacillus mojavensis ST-3, and Bacillus megaterium ST-4 were non-symbiotically isolated from the heamolymph of dead Steinernema-infected Galleria mellonella larvae and molecularly characterized. The bacterial cells and filtrates from P. alcaligenes ST-1 strongly inhibited the growth of Staphylococcus aureus through disk diffusion (43 mm), minimum inhibitory concentration (2.5 µL/mL), and minimal bactericidal concentration (5 µL/mL) assay. Conclusively, the direct application of endogenous Steinernema-associated EPB as an antibacterial agent for antibiotic-resistant bacteria looked promising.