Two new cytochalasans flavichalasine N (1), flavichalasine O (2), together with six known cytochalasans (3–8), were isolated from Aspergillus flavipes PJ03-11 through the application of OSMAC (one strain many compounds) strategy. Flavichalasine O (2) represented the first example of cytochalasans possessing a nitrogen-oxygen heterocycle at the macrocyclic ring part. Their structures were established on the base of extensive spectroscopic analysis. Compounds 1–4 exhibited significant cytotoxic activities against three human cancer cell lines (THP1, HL-60 and PC3) with IC50 values ranging from 3.00 to 15.10 lM.