Five new cytochalasins, Z16–Z201–5, and three known ones, 6–8, were isolated from Aspergillus flavipes, an endophytic fungus associated with Acanthus ilicifolius.Their structures were elucidated on the basis of comprehensive spectral analysis. Cytochalasin Z172 and rosellichalasin 8 showed cytotoxic activities against A549 cell lines with IC50values of 5.6 and 7.9μM, respectively.