Two new antimicrobial peptides belonging to brevinin 1 (B1) and brevinin 2 (B2) families were identified from the skin secretion of Indosylvirana aurantiaca, an endemic frog of Western Ghats, India, through shotgun cloning. Antibacterial, antibiofilm and cytotoxic effects of these peptides were evaluated and compared with their C-terminally amidated forms. Both the amidated peptides (B1-NH2 and B2-NH2) showed significantly enhanced broad-spectrum activities against Grampositive and Gram-negative bacteria and the latter was found to have potent biofilm inhibition properties against Klebsiella pneumoniae over other peptides. Both forms of B2 were less cytotoxic against human red blood cells (hRBC) and peripheral blood mononuclear cell (PBMC). Hence, B2 and its amidated analog have good therapeutic value and these peptides could be considered as potential lead molecules for further development.