Pyrenocines are better known as phytotoxic metabolites been found in Alternaria, Curvularia, Penicillium and Phomopsis strains. The class of compounds showed several biological activities, reportedly being phytotoxic, antifungal, cytotoxic, antitrypanosomal and antimalarial.1, 2, 3 Fungi of the genus Colletotrichum, a class of the widely distributed endophytic fungi, were reported as prolific producers of bioactive metabolites.4, 5, 6, 7 However, there are few reports on pyrenocines found in genus Colletotrichum. During our ongoing chemical investigations of endophytic fungi as sources of new cytotoxic natural products, the organic solvent extract of solid substrate fermentation culture of a Colletotrichum sp. HCCB03289 strain displayed cytotoxicity against cancer cell lines. Bioassay-guided fractionation of the extract led to the isolation of two novel pyrenocines N–O (1–2), and five known pyrenocines macommelin-9-acetate (3),8 pyrenocine A (4),9 pyrenocine B (5),9 pyrenocine E (6)10 and novae-zelandin A (7).11 Details of the isolation, structure elucidation and the cytotoxic activities of these compounds are presented here.