The present study aimed to screen the in vitro antimicrobial activity of ethyl acetate fraction isolated from the leaves of Heterophragma adenophyllum against Escherichia coli, Salmonella enterica, Staphylococcus aureus, Bacillus anthracis and Klebsiella pneumonia broad spectrum microorganism. The study was carried out by using standard nutrient broth for microbes and streptomycin was taken as a positive control. Ethyl acetate fraction used as a test sample and DMSO as a negative control. From the results of the zone of inhibition it was concluded that the ethyl acetate fraction was pusses in vitro antimicrobial activity while results of minimum inhibitory concentration, it was revealed that all bacterial strains were sensitive towards selected fraction for the study.