Pteris vittata, commonly known as ‘Brake Fern’, is a relatively uninvestigated species as far as antimicrobial activity is concerned. Different aqueous methanolic (70, 80, and 90%) extracts of P. vittata were tested for the growth of eight intestinal microorganisms, by using disc diffusion and micro-dilution methods as recommended by NCCLS. The 70% aqueous methanolic extract showed potent activity against Pseudomonas aeruginosa and Klebsiella pneumoniae. Total phenol content of the plant showed a substantial amount of phenols (0.97%); in addition the flavonoid rutin was identified by HPLC and MS. The present investigation is the first biological report in fern species P. vittata ascertaining the antimicrobial activity; the antimicrobial activity of rutin against the above microorganisms has also been shown.