Xenorhabdus and Photorhabdus are members of the family Enterobacteriaceae, and are engaged in a mutualistic association with the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. In the present study, the organic fraction of ethyl acetate bacterial crude extracts of the symbiotic bacteria, Xenorhabdus stockiae and Photorhabdus luminescens isolated from entomopathogenic nematodes, were characterized by High Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared Spectroscopic (FTIR) methods, and evaluated for their antibacterial activity on selected pathogenic bacteria, Escherichia coli, Staphylococcus aureus, Salmonella typhi and Klebsiella pneumonia by agar well plate method. When compared to X. stockiae, P. luminescens showed higher antibacterial activity at maximum concentration on K. pneumonia and S. aureus, respectively. FTIR analysis of the crude ethyl acetate extract of X. stockiae showed the corresponding peaks values of 2373.33 and 544.93 cm-1 whereas P. luminescens showed the presence of peaks at 2128.00 and 552.80 cm-1, respectively indicating the functional group of P-H and isocyanides stretching. The present study suggests that the antibacterial activity might be due to the effective compounds from the symbiotic bacteria of entomopathogenic nematodes, and that they could be used as bioinsecticides in the future.