The fungus Talaromyces wortmannii, isolated from a soil sample collected in China's Yunnan province, produced four novel 22-membered macrolides, namely, wortmannilactones A-D (1-4). Structures 1-4 were elucidated by extensive 1D and 2D NMR and MS spectral analyses. Compounds 1-4 exhibited in vitro cytotoxic activity against several human cancer cell lines with IC50 values ranging from 28.7 to 130.5 microM.