We report new stromatal azaphilone pigments rubiginosins Z-X from the ascomycete Hypoxylon rubiginosum, as well as rubiginosins Z and W from H. texense, which were isolated along with known monomeric and dimeric congeners. Structures were elucidated using comprehensive HRMS, NMR, and ECD analysis, revealing azaphilones from both fungi to be exclusively C-8(S)-configured. The orsellinic acid (OA)-carrying rubiginosins A, Z and dimeric rutilins A-B exhibited cytotoxicity. Rubiginosins X-W bearing linear polyketide side chains as well as rutilins A-B were antimicrobial. Structures of the differently-substituted azaphilones were linked to two putative biosynthetic gene clusters (BGCs; hraza1/2) in H. rubiginosum, which are proposed to collaboratively synthesize the OA-substituted azaphilones. These share high homology with the azaphilone-forming BGCs hfaza1/2 from H. fragiforme. Comparison of hraza and hfaza suggests that lack of an FAD-dependent monooxygenase and acyltransferase gene in hraza1 prevent formation of C-8(R)-configured fatty acid-substituted azaphilones in H. rubiginosum. The polyketide synthase-derived side chain of rubiginosins C and X-W is not encoded in the respective BGCs, showing that a third BGC is hypothetically involved in their formation. Cross-interaction of three BGCs which are forming a single molecule is unprecedented in fungal natural product biosynthesis.