Introduction: Foodborne disease is a serious public health problem worldwide and most of the disease is related to food spoilage by bacteria. Thus, there is a need for the discovery of a natural antibiotic to inhibit these foodborne bacteria. Materials and Methods: Disk diffusion and broth microdilution assays were performed to access the antibacterial activity of the fungal extracts against test bacteria. Meanwhile, the scanning electron microscope (SEM) was employed to study the structural degeneration of bacterial cells exposed to the extracts. Results: The ethyl acetate extract of Lasiodiplodia pseudotheobromae IBRL OS-64 demonstrated favorable antibacterial activity towards all test bacteria with inhibition zone ranged from 15.0 mm to 26.0 mm. All test bacteria were resistant to hexane and butanolic extracts. Minimum inhibition concentration (MIC) values of the ethyl acetate extract between 62.5 µg/mL to 500 µg/mL whereas minimum bactericidal concentration (MBC) was in the range of 125 µg/mL to 2000 µg/mL. The SEM micrographs exhibited major abnormalities that occurred on the bacterial cells after exposure to the extract with alteration in their morphology and cell death beyond repair. Conclusion: The extracts of L. pseudotheobromae IBRL OS-64 may be an effective antibacterial agent to treat foodborne bacteria.