Bacterial fruit blotch (BFB) is a bacterial disease that devastates Cucurbitaceae crops worldwide, causing significant economic losses. Currently, there is no means to treat or control the disease. This study focused on exploring the antibacterial properties of endophytic fungi against Acidovorax citrulli (Aac), the causative agent of BFB. Based on disc diffusion, time kill and MIC microdilution broth assays, four endophytes showed promise in controlling Aac. Nonetheless, only one strain, Neocosmospora sp. MFLUCC 17-0253, reduced the severity of disease on watermelon and melon seedlings up to 80%. Structure analysis revealed production of several compounds by the fungus. Three of these secondary metabolites, including mixture of 2-methoxy-6-methyl-7-acetonyl-8-hydroxy-1,4-maphthalenedione and 5,8-dihydroxy-7-acetonyl-1,4-naphthalenedione, anhydrojavanicin, and fusarnaphthoquinones B exhibited antagonistic activity against Aac. The chemical profile data in planta experiment analyzed by LC-Q/TOF-MS suggested successful colonization of endophytic fungi in their host plant and different metabolic profiles between treated and untreated seedling. Biofilm assay also demonstrated that secondary metabolites of Neocosmospora sp. MFLUCC 17-0253 significantly inhibited biofilm development of Aac. To the best of our knowledge, secondary metabolites that provide significant growth inhibition of Aac are reported for the first time. Thus, Neocosmospora sp. MFLUCC 17-0253 possesses high potential as a biocontrol agent for BFB disease.