<jats:title>Abstract</jats:title><jats:p>Three new prenylated 2,5‐diketopiperazines, namely neofipiperazines A–C (<jats:bold>1</jats:bold>–<jats:bold>3</jats:bold>, resp.), were isolated from the culture of <jats:italic>Neosartorya fischeri</jats:italic> CGMCC 3.5378, together with six known ones. The structures of the new compounds were elucidated by comprehensive spectroscopic analysis, especially HR‐ESI‐MS and NMR experiments. All the prenylated diketopiperazines, except <jats:bold>7</jats:bold>, were isolated for the first time from the species.