Five cyclopenta [d]pyrano [4,3-b]pyran-1,7(6H)-dione 6/6/5-fused tricyclic ring system containing metabolites peniapyrones A−E (1−5), and four previously undescribed cyclopenta [4,5]furo [3,2-c]pyran-1-one 6/5/5-fused tricyclic ring system containing compounds peniapyrones F−I (6−9), were isolated from the endophytic Penicillium brefeldianum F4a. Their structures, including absolute configurations, were determined through spectroscopic analysis and quantum chemical calculations. Peniapyrones D (4) and E (5) were a pair of diastereoisomers. Compounds 1, 3, and 5−9 showed cytotoxic activity against AsPC-1, CRL-2234, and MCF-7 cancer cell lines. Compounds 1, 3, 6, 8, and 9 inhibited the Kirsten rat sarcoma viral oncogene homologue (KRAS) mutant AsPC-1 cell line.