Mangrove endophytic fungi are recognized as prolific sources of structurally diverse and bioactive natural products. In this study, 19 cultivable endophytic fungal strains were isolated and taxonomically characterized from mangrove plant tissues collected at the Shankou Mangrove Nature Reserve (Guangxi, China). One isolate, Aspergillus sclerotiorum BM19, exhibited broad-spectrum inhibitory activity against multiple aquatic pathogenic Vibrio strains. Bioactivity-guided fractionation of its ethyl acetate crude extract led to the isolation of penicillic acid (1), whose structure was fully elucidated by 1D/2D NMR and HRESIMS. Penicillic acid showed potent anti Vibrio parahaemolyticus activity with a minimum inhibitory concentration (MIC) of 6.25 μg/mL and displayed no obvious cytotoxicity toward human HaCaT keratinocytes. Growth curve assays revealed a concentration-dependent antibacterial effect: 0.5 × MIC delayed logarithmic growth, whereas 1 × and 2 × MIC completely suppressed bacterial proliferation. Transmission electron microscopy (TEM) visualized cellular distortion, compromised cell envelopes, and cytoplasmic leakage at MIC, with severe envelope disruption at 2 × MIC. Confocal laser scanning microscopy (CLSM) combined with SYTO 9/PI double staining further confirmed the concentration-dependent impairment of cell envelope integrity, as evidenced by reduced green fluorescence (intact cells) and increased red fluorescence (damaged cells). Collectively, these findings indicate that penicillic acid inhibits V. parahaemolyticus and is associated with concentration-dependent impairment of cell-envelope integrity. Although penicillic acid is a known mycotoxin, its selective activity against pathogenic vibrios and low toxicity to human keratinocytes highlight its potential as a lead compound for aquaculture applications. Nevertheless, further systematic evaluation of in vivo efficacy and biosafety is required prior to translational development.