<jats:title>Abstract</jats:title><jats:p>Five new cytochalasins, Z<jats:sub>16</jats:sub>–Z<jats:sub>20</jats:sub> (<jats:bold>1</jats:bold>–<jats:bold>5</jats:bold>), and three known ones, <jats:bold>6</jats:bold>–<jats:bold>8</jats:bold>, were isolated from <jats:italic>Aspergillus flavipes</jats:italic>, an endophytic fungus associated with <jats:italic>Acanthus ilicifolius.</jats:italic> Their structures were elucidated on the basis of comprehensive spectral analysis. Cytochalasin Z<jats:sub>17</jats:sub> (<jats:bold>2</jats:bold>) and rosellichalasin (<jats:bold>8</jats:bold>) showed cytotoxic activities against A‐549 cell lines with <jats:italic>IC</jats:italic><jats:sub>50</jats:sub> values of 5.6 and 7.9 μ<jats:sc>M</jats:sc>, respectively.