Nature is a generous source of compounds with the potential for prevention of infections. Antimicrobial screening of aqueous extract from bark of wild Himalayan cherry (Prunus cerasoides) was carried out against various pathogenic microorganisms with inhibition zone ranging from 19 to 24 mm. An optimization strategy, which included classical method and statistical method (RSM), was applied to optimize the effect of process variables. Fifteen percent plant material extracted at 40 A degrees C for 60 min and at its natural pH (4.5) exhibited best antimicrobial activity with an average zone of inhibition ranging from 19 to 29 mm. Statistical optimization using RSM further enhanced the activity by 1.09-1.24 folds. Minimum inhibitory concentration of the aqueous extract against different microorganism ranged from 1 to 10 mg/ml. The aqueous extract was found to be reasonably thermostable at boiling temperature for 1 h. Viable cell count (VCC) studies of the extract showed it to be bactericidal in nature. Further, the aqueous extract was found to be neither cytotoxic nor mutagenic, when evaluated by MTT assay and Ames mutagenicity test. The results suggest that the aqueous extract of P. cerasoides could be a potential source to obtain new antimicrobials and effective herbal medicines to combat the problem of ever emerging microbial resistance.