Abstract: The focal theme of the present investigation is to explore the antimicrobial activity of Tannery Fleshing (TF) utilizing Labeo rohita gut bacteria against common microbial pathogens. The study involves isolation of the bacteria through culture technique, biochemical characterization, Scanning Electron Microscopic and phylogenetic cluster analysis. Antimicrobial activities were determined through agar diffusion technique. Partial 16S-rRNA gene sequencing analysis shows that the isolate exhibited homology with Bacillus megaterium. Among the bacterial pathogens maximum inhibition was observed against Salmonella typhi and Bacillus subtilis when compared to standard antibiotic followed by Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris. Minimum inhibitory effect was observed with Staphylococcus aureus and Pseudomonas aeruginosa. Most susceptible fungal pathogens include Microsporum canis and Aspergillus fumigatus whereas Trichophyton mentagrophytes and Penicillium chrysogenum were least susceptible. Antifungal activity of Bacillus megaterium remained almost similar to standard drug in Fusarium moniliforme, Aspergillus niger, Candida albicans. The in vitro studies indicate that Bacillus megaterium ANFLR1 isolated from Labeo rohita can inactivate pathogenic Gram-positive and Gram-negative bacteria as well as fungi and can be of potential use as an antibiotic and probiotic.