Monotheca buxifolia has traditionally been employed in folk medicines to cure of infectious diseases. Current study was aimed to standardize the M. buxifolia leaves extract and evaluate its antibacterial and anticancer activity. Phytochemical analysis was carried through GC, GC/MS, FTIR, and ICP-OES analytical techniques. Antibacterial assay of the crude extract was performed by using tetrazolium micro plates. The extract treated bacteria were observed under (AFM) atomic force microscope and PCR was used for DNA amplification. The anti-proliferative activity of M. buxifolia leaves extract was examined through MTT cytotoxicity assay. The bacterial strains employed in this study were S. epidermidis ATCC (13518), S. aureus ATCC (25923), P. aeruginosa ATCC (10145), and E. coli ATCC (10536). Minimum inhibitory concentration (MIC50) against gram positive bacteria was significantly (p<0.01) achieved at 50 and 75μg/mL. MIC50 against E. coli and P. aeruginosa was also significant at 100μg/mL (p<0.01). M. buxifolia leaves extract damaged the cell walls gram-positive and gram-negative bacteria, while biofilm around gram positive bacteria was significantly damaged. The DNA decantation was also inhibited of S. aureus and S. epidermidis, however, no any impact was observed on E. coli and P. aeruginosa DNA decantation. The cytotoxicity findings suggested that the crude extract of M. buxifolia leaves at 1000μg/mL gives significant inhibition 73.96±2.0%, 83.76±1.2%, 77.66±1.2% and 72.67±1.6% against MDA-MB-231, MCF-7, HeLa and H460 cell lines respectively at (p<0.001). It may be concluded that M. buxifolia leaves extract have significant and promising antibacterial and anti-cancer activities which could be helpful to establish new antimicrobial and anticancer agents.