Prunus cerasoides is a traditionally well known for human health in various ways and particularly its bark is reported to possess high therapeutic applications in wound healing, foot and mouth disease, and indigestion etc. But there is scanty literature available on its systematic studies and phytoconstituents responsible for antimicrobial activity so the work is proposed. The main aim of this study is to reveal the phytoconstituents responsible for antimicrobial and antibiofilm action to demonstrate the effectiveness of such compounds by extrapolating the data using clinical isolates of pathogenic bacteria. In the present study, evaluation of P. cerasoides organic extract and phytoconstituents for their antimicrobial and antibiofilm potential against reference microbial strains was carried out. Antimicrobial potential was carried out using agar diffusion assay and biosafety of organic extract and its phytoconstituents was evaluated by MTT and Ames mutagenicity assay. Ethyl acetate was found to be the best organic extractant, where Klebsiella pneumoniae 1 (39.5mm) and Staphylococcus aureus (22.5mm) were the most sensitive microorganisms, respectively. Among the major phytoconstituents, flavonoids (14.5-33.5mm), diterpenes (14-28.7mm), and cardiac glycosides (11.5-20.5mm) exhibited broad-spectrum antimicrobial activity. Ethyl acetate extract showed better potency with lowest minimum inhibitory concentration (0.1-10mg/ml) than the most active partially purified phytoconstituents (0.5-10mg/ml). Total activity potency for ethyl acetate extract ranged from 26.66-2666ml/g and for flavonoids, it was 41-410ml/g, thus considered as highly potent and bactericidal in nature as evidenced from VCC study. The major bioactive compounds were found to be biosafe. The most active phytoconstituents were found to have antibiofilm potential, as well as effective against clinical isolates of MRSA, thus, the findings indicate that P. cerasoides stem bark could be a potential source for development of broad-spectrum drugs against multidrug-resistant bugs.