There is an urgent call to search for novel natural compounds against developing multidrug-resistant microorganisms. The present work focuses on the characterization of a plant-associated fungus having bioactivity against methicillin-resistant Staphylococcus aureus (MRSA) strains. A fungal strain P31 was isolated from bark of Dillenia pentagyna and identified as Pestalotiopsis microspora. The maximum anti-MRSA activity was observed from extract of P31 grown in sabouraud dextrose broth. The minimum inhibitory concentrations(MIC) values of P31 extract were 14 μg/ml for methicillin-sensitive S. aureus (MSSA) and 32 μg/ml for MRSA strain, respectively. A crude P31 extract showed strong bactericidal activity by killing all treated MRSA cells within 24 h of treatment at their respective MIC value. A scanning electron microscopic study visualized morphological damage of MRSA cells. The membrane permeability of P31 extract-treated MRSA cells gradually increased which caused release of internal cytoplasmic nucleic acids, proteins and potassium ions (K+ ) from cells suggesting cell lysis or leakage from cells. A very low concentration of P31 extract was able to inhibit biofilm formed by MRSA cells. Thin layer chromatographic separation followed by gas chromatography-mass spectrometry analysis of the P31 extract revealed a number of antimicrobial compounds along with an anti-MRSA compound 2,4-di-tert-butylphenol. In addition, the P31 extract also showed in-vitro human blood clot lysis activity at various concentrations. The clot lysis activity of P31 extract was found maximum at 500 µg/ml. These findings suggest that fungal isolate P31 has potential as a source of anti-MRSA compounds useful in staph infections.