The entomopathogenic nematodes (EPNs) are a nematode-bacterium complex. The symbiotic nematode-bacterial mutualism is highly specific. Novel EPN belonging to the family rhabditidae were reported for the first time from the Central Tuber Crops Research Institute. These new EPNs belonging to the Rhabditis sp. and their symbionts offer great scope for their exploitation of bio separation and identification of novel bioactive molecules. The present work is mainly focused on the antimicrobial activity of a new entomopathogenic bacteria Acinetobacter. The bacteria were isolated from infective third stage dauer juveniles of the nematode isolate collected from Vellayani, Thiruvananthapuram. The incubation time of the bacteria was standardized and cell free culture filtrate was prepared. The cell free culture filtrate was then separated into an organic and aqueous fraction. Antibacterial and antifungal activity of the organic fraction were tested. The organic fraction of 72 hr have highest antibacterial activity against B.subtilis with a zone diameter of 20 mm and lowest antibacterial activity with a zone diameter of 13 mm against P. aeruginosa and antifungal activity (29 mm zone diameter) in case of A. flavus and lower in case of F. oxysporum (18 mm zone diameter). Antimycotic and antibacterial activity was not observed in 24 hr organic fraction. From the study, it can be concluded that the organic fraction has significant antibacterial and antifungal activity. The present study also reveals that this entomopathogenic bacteria will be useful for the production of bioactive metabolites effective against bacterial and fungal diseases of plants and animals.