Objective:To determine the antibacterial, antifungal, minimum inhibitory concentration (MIC) and the protease activity of the stingray mucus Dasyatis sephen (D. sephen) and Himantura gerrardi (H. gerrardi). Methods: Antimicrobial activity of crude aqueous, acidic and organic mucus extract was evaluated by disc diffusion method against human pathogens, MIC of the active samples were determined by spectrophotometric method and the protease activity which is responsible for the antimicrobial activity was determined by using zymogram method. Results:The crude acidic extracts of both the species showed antibacterial activity against Salmonella typhi (S. typhi), Klebsiella pneumoniae, Streptococcus aureus, Escherichia coli (E. coli), Vibrio cholerae (V. cholerae) and the acidic extracts of both the species exhibit antifungal activity against all the tested pathogens. Remaining extracts didn't show any inhibitory activity. The acidic extracts of H. gerrardi is significantly active against S. typhi, E. coli, V. cholerae, Trichophyton mentagrophytes (T. mentagrophytes), Alternaria alternaria (A. alternaria), Trichophyton rubrum (T. rubrum), Candida tropicalis (C. tropicalis) at the minimum concentration of 16μg/mL, but the acidic extract of D. sephen required 32μg/mL of protein to inhibit S.typhi, E. coli, Aspergillus niger (A. niger), penicillium sp, T. mentagrophytes, A. alternaria. Both the D. sephen and H. gerrardi shows the proteolytic activity above the molecular mass of> 66 KDa. The characterization of protease class using inhibitors showed the presence of both serine and metallo protease in the the samples. Conclusions:Protease activity present in the sting ray mucus is one of the key factor responsible for the antimicrobial activity and the results proved the role of mucus in the innate immunity.