Background and Objectives: Endophytic fungi are believed to possess compounds as antibacterial agents. This study was designed to determine in vivo antibacterial activity of the crude extracts from Lasiodiplodia pseudotheobromae IBRL OS-64 against pathogenic bacteria. Materials and Methods: The qualitative and quantitative screenings were performed using agar plug and disk diffusion antimicrobial tests, respectively. Besides that, the MIC and MBC value of the extracts were determined using broth microdi- lution assay and morphological changes of the bacterial cells exposed to the extract were observed under Scanning Electron Microscope (SEM). Results: Agar plug diffusion assay revealed that V. parahaemolyticus ATCC 17802 and Exiguobacterium profundum IBRL MA6 were the most sensitive to the extract with the size of inhibition zones of 11 to ≤ 20 mm. The MIC and MBC values of the extract varied depending on the test bacteria. Observation through SEM revealed that the bacterial cells exposed to the extract experienced severe damage such as irregular shape with crumpled and shrunken cells which led to cell death. Conclusion: The data suggest that the crude extracts of L. pseudotheobromae IBRL OS-64 exert antibacterial activity against test bacteria and principally affect the cell wall in growing pathogenic bacterial cells.