Abstract Background Endophytic fungi play a vital role in plant defense system by secreting various antimicrobial agents. To evaluate the antimicrobial activity of the endophytic fungi of the mangrove plant Heritiera fomes (Buch. -Ham), plants were collected from the Sundarbans, Bangladesh. The endophytic fungi were subsequently isolated and identified. Results Fifty-five endophytic fungi were isolated from the leaves, root, and bark of H. fomes (Buch. -Ham). Genomic DNA was extracted for PCR (polymerase chain reaction) by specific primers to multiply ITS region and sequences were determined. Nucleotide sequence data were submitted to the Genbank and accession number for each fungal strain was obtained. Antimicrobial activity of the ethyl acetate (EtOAc) and methanolic extracts of eleven species from both fermentation and mycelium, respectively, were analyzed by microtiter plate-based antimicrobial assay incorporating resazurin as an indicator of cell growth against two Gram-positive bacteria namely Staphylococcus aureus NCTC 12981 and Micrococcus luteus NCTC 7508, two Gram-negative bacteria namely Escherichia coli NCTC 12241 and Pseudomonas aeruginosa NCTC 12903, and a fungus Candida albicans ATCC 90028. All the endophytic fungal extracts exhibited antimicrobial activities against more than one-tested pathogenic microbial strains. Overall, methanolic extracts showed greater activity than EtOAc extracts. Pseudopestalotiopsis camelliae-sinensis, Pestalotiopsis microspora, and Penicillium copticola were the most active endophytic fungal strains and exhibited strong inhibitory activity against the microorganisms under investigation and their MIC values ranged from 0.0024 to 5.0 mg/mL. Methanolic extracts of both P. camelliae-sinensis and P. microspora showed the highest antibacterial activity (MIC value of 0.0024 mg/mL) against P. aeruginosa NCTC 12903. Conclusion This study showed that the isolated and identified endophytic fungi from H. fomes (Buch. -Ham) could be potential sources of antimicrobial agents.