Three new N-methyl-4-hydroxy-2-pyridinone analogues, 6-epi-oxysporidinone (3), the dimethyl ketal of oxysporidinone (4), and N-demethylsambutoxin (5), along with the known compounds (-)-oxysporidinone (1), (-)-sambutoxin (2), wortmannin (6), enniatin A (7), enniatin A1 (8), and enniatin B1 (9) were isolated from Fusarium oxysporum (N17B) by bioassay-guided fractionation. Compounds 1 and 3 showed selective fungistatic activity against Aspergillus fumigatus, and wortmannin had selective potent activity against Candida albicans. Moderate activity was observed with the enniatins 7-9 against C. albicans, Cryptococcus neoformans, and Mycobacterium intracellulare. Compounds 1-5 had no activity against the agriculturally important fungi Fusarium verticillioides (syn. F. moniliforme) and Aspergillus flavus.