As part of research on bioactive metabolites from marine-derived fungi, the marine-derived fungus Diaporthaceae sp. PSU-SP2/4 isolated from a marine sponge collected from Pak Meng beach, Sikao, Trang province, Thailand was chemically investigated. Purification of the mycelial EtOAc extract by different chromatographic techniques led to the isolation of one new pentacyclic lactam, diaporthalasin (1). The broth EtOAc extract yielded one new ethyl trihydroxytridecatrienoate derivative, diaporthacol (2, isolated as its triacetate derivative 3), together with five known compounds (R-mevalonolactone, dothiorelone C, (4S,7S,13S)-4,7-dihydroxy-1,3-tetradeca-1,5-dienolide, 4β-acetoxy-9β,10β,15α-trihydroxyprobotrydial, O-methyldihydrobotrydial). The structures were established by spectroscopic techniques (especially 1D and 2D NMR data). Compound 1 displayed significant antibacterial activity against both Staphylococcus aureus and methicillin-resistant S. aureus with equal MIC values of 2 mg/mL. Compound 8 was weakly active against both strains, while compounds 6 and 7 were inactive. Compound 6 was mildly active toward MCF-7 cancer cells, and both 6 and 7 were non-cytotoxic to noncancerous Vero cells.