Background Foraminispora rugosa is a species reported from Brazil, Venezuela, French Guiana, Costa Rica and Cuba. It is a basidiomycete in the Ganodermataceae family. In this study, both chemical composition and cytotoxicity of the ethanolic extract of F. rugosa were investigated for the first time. Results Phylogenetic analysis confirmed the identification of the specimens, and the results of cytotoxicity assays showed that at concentrations of 7.8–500.0 µg/mL the ethanolic extract displayed weak cytotoxicity against the tested cell lines. Five oxylipins were identified by ultra high performance liquid chromatography coupled with quadrupole time-of-flight and mass spectrometry (UHPLC-QTOF–MS). Conclusions This study provides new insights into the current knowledge of bioactive compounds produced by macrofungi, and provides data for future biological assays with relative selectivity and safety.