To examine the antibacterial activity of ethyl acetate, ethanol and methanol extracts of Azadirachta indica (A. indica) on fish pathogens viz., Aeromonas veronii, Aeromonas hydrophila, Acinetobacter junii, Acinetobacter tandoii, Acinetobacter spp. and Pseudomonas stutzeri isolated from diseased blackspot barb, Dawkinsia filamentosa. The naturally infected fish, D. filamentosa were collected from Mettur Dam and the associated bacteria in the ulcerative lesions were isolated, sequenced the amplified product and tentatively identified through sequence similarity. The leaves powder was extracted successively with 250 ml of ethyl acetate, ethanol and methanol solvents by using Soxhlet extractor for 8 h at a temperature not exceeding the boiling point of the solvent. Ethyl acetate, ethanol and methanol extracts were subjected to phytochemical analysis as well as the antimicrobial assay using agar well diffusion method. Totally six fish bacterial pathogens were isolated from the ulcerative of lesions blackspot barb fish (D. filamentosa). All the gene sequences were deposited in the NCBI and received their accession numbers. The isolated strains were: A. veronii (KX688046), P. stutzeri (KX721473), A. hydrophila (KX756709), A. spp. (KX775221), A. junii (KX756708) and A. tandoii (KX775222). The different crude extracts of A. indica showed significant inhibitory effects on all the isolates. In which, the ethyl acetate extract showed maximum zone of inhibition on against P. stutzeri (23 mm) A. junii (22 mm), A. hydrophila (19 mm) and A. tandoii (18 mm). The ethanol extract showed moderate zone of inhibition on A. junii (15 mm) and A. veronii (14 mm) and also the methanol extract on A. junii (15 mm) and A. veronii (14 mm). The present study revealed that the crude extracts A. indica leaves possesses significant antibacterial activity on isolated fish pathogens. The susceptibility test was conducted on isolates using antibiotics, selected based on their importance to human medicine and on fish production. To conclude the present findings the ethyl acetate leaf extracts could be used as potential sources of antimicrobial agents.