A chemical investigation of the filamentous fungus Aspergillus californicus led to the isolation of a polyketide-nonribosomal peptide hybrid, calipyridone A (1). A putative biosynthetic gene cluster cpd for production of 1 was next identified by genome mining. The role of the cpd cluster in the production of 1 was confirmed by multiple gene deletion experiments in the host strain as well as by heterologous expression of the hybrid gene cpdA inAspergillus oryzae. Moreover, chemical analyses of the mutant strains allowed the biosynthesis of 1 to be elucidated. The results indicate that the generation of the 2-pyridone moiety of 1 via nucleophilic attack of the iminol nitrogen to the carbonyl carbon is different from the biosynthesis of other fungal 2-pyridone products through P450-catalyzed tetramic acid ring expansions. In addition, two biogenetic intermediates, calipyridones B and C, showed modest inhibition effects on the plaque-forming ability of SARS-CoV-2.