<jats:title>Abstract</jats:title><jats:p>From the <jats:italic>n</jats:italic>‐hexane fraction of the fruits of <jats:italic>Vitex agnus‐castus</jats:italic>, two labdane‐type diterpenes, vitetrifolin B and C, were isolated by means of multiple chromatographic separations, together with the previously identified rotundifuran, vitexilactone and the sesquiterpene spathulenol. From the EtOAc fraction, eupatorin was identified for the first time, besides the known casticin, penduletin, vitexin and orientin. The <jats:italic>n</jats:italic>‐hexane, EtOAc and MeOH–H<jats:sub>2</jats:sub>O fractions of the MeOH extract of <jats:italic>Agni‐casti fructus</jats:italic> were subjected to <jats:italic>in vitro</jats:italic> antioxidant assays. The EtOAc extract displayed a significant concentration‐dependent effect when tested by 1,1‐diphenyl‐2‐picrylhydrasyl (DPPH) free radical assay (IC<jats:sub>50</jats:sub> = 68 µg/mL) and against the autooxidation of a standard rat brain homogenate (IC<jats:sub>50</jats:sub> = 14 µg/mL). The MeOH–H<jats:sub>2</jats:sub>O fraction was less active with 3643 µg/mL (DPPH test) and IC<jats:sub>50</jats:sub> = 125 µg/mL (rat brain homogenate), while the <jats:italic>n</jats:italic>‐hexane phase proved to be inactive. The main flavonoid constituents of the EtOAc extract, casticin, vitexin and orientin were assayed for antioxidant activity and found that only casticin possesses a marked lipid peroxidation inhibitory effect (IC<jats:sub>50</jats:sub> = 0.049 m<jats:sc>m</jats:sc>) compared with that of the positive control ascorbic acid (IC<jats:sub>50</jats:sub> = 0.703 m<jats:sc>m</jats:sc>). Copyright © 2007 John Wiley & Sons, Ltd.