Fungi have become an invaluable source of bioactive natural products, with more than 5 million species of fungi spanning the globe. Fractionation of crude extract of Neodidymelliopsis sp., led to the isolation of a novel polyketide, (2Z)-cillifuranone (1) and five previously reported natural products, (2E)-cillifuranone (2), taiwapyrone (3), xylariolide D (4), pachybasin (5), and N-(5-hydroxypentyl)acetamide (6). It was discovered that (2Z)-cillifuranone (1) was particularly sensitive to ambient temperature and light resulting in isomerisation to (2E)-cillifuranone (2). Structure elucidation of all the natural products were conducted by NMR spectroscopic techniques. The antimicrobial activity of 2, 3, and 5 were evaluated against a variety of bacterial and fungal pathogens. A sodium [1-13C] acetate labelling study was conducted on Neodidymelliopsis sp. and confirmed that pachybasin is biosynthesised through the acetate polyketide pathway.