The antimicrobial potentiality of Polyalthia longifolia seed oil and its fatty acid methyl ester (FAME) extract was evaluated against multi drug resistant strains of bacteria and fungi of clinical origin. A sensitivity test was performed to determine the resistance pattern for marketed antimicrobial agents against the selected microorganisms. The results of antimicrobial assay revealed that P. longifolia seed oil and its FAME extract were effective against all the microorganisms studied. Enterococcus faecalis, Streptococcus mutans and Streptococcus bovis were observed to be the most susceptible strains. Among the tested Gram-negative bacteria, Klebsiella pneumoniae was found to be the most sensitive, while Salmonella typhimurium was the most resistant bacteria. In the case of Gram-positive bacteria, E. faecalis was the most sensitive, while S. aureus was the most resistant strain. The Candida albicans was found to be highly sensitive to the action of FAME extract with the least minimum inhibitory concentration (MIC) of 19.5 µg/ml. The MIC values were lower than MBC/MFC values suggesting that the P. longifolia seed oil and its FAME extract inhibit the growth of test organisms while being bactericidal/fungicidal at higher concentrations. In addition, the physiochemical properties and fatty acid composition of P. longifolia seed oil were also investigated. Thus, it was found that P. longifolia seed oil offers potential antibacterial property against the reference strains and could be a possible source to obtain new and effective herbal medicines to treat infections caused by multi-drug resistant strains of microorganisms from community as well as hospital settings.