Epicoccum nigrum produces epipyrone A (orevactaene), a yellow polyketide pigment. Its biosynthetic gene cluster was previously characterized in E. nigrum KACC 40642. The YES liquid culture of this strain revealed high-level production of epicoccamide (EPC), with an identity that was determined using liquid chromatography-mass spectrometry analysis and molecular mass search using the SuperNatural database V2 webserver. The production of EPC was further confirmed by compound isolation and nuclear magnetic resonance spectroscopy. EPC is a highly reduced polyketide with tetramic acid and mannosyl moieties. The EPC structure guided us to localize the hypothetical EPC biosynthetic gene cluster (BGC) in E. nigrum ICMP 19927 genome sequence. The BGC contains genes encoding highly reducing (HR)-fungal polyketide synthase (fPKS)-nonribosomal peptide synthetase (NRPS), glycosyltransferase (GT), enoylreductase, cytochrome P450, and N-methyltrasnferase. Targeted inactivation of the HR-fPKS-NRPS and GT genes abolished EPC production, supporting the successful localization of EPC BGC. This study provides a platform to explore the hidden biological activities of EPC, a bolaamphiphilic compound.