Periconiasins A-C (1-3), new cytochalasans with an unprecedented 9/6/5 tricyclic ring system, were isolated from the endophytic fungus Periconia sp. F-31. Their molecular structures and relative configurations were unambiguously established on the basis of extensive spectroscopic data analysis. The absolute configurations were determined by analyzing the single-crystal X-ray diffraction data of one of the esterified derivatives of 1 (1a) and circular dichroism (CD) spectroscopic data. The biosynthetic pathway of 1-3 was proposed to be typical of fungal PKS-NRPS hybrids formed from one acetyl-CoA starter and six malonyl-CoA extenders coupled with one leucine-CoA. Compounds 1 and 2 showed significant cytotoxicity against human HCT-8 cancer cells.