Grifola frondosa, a medicinal and edible mushroom, was cultivated on substrates composed of olive oil press cakes with different supplements. Crude extracts were prepared from mature fruiting bodies and tested for their capacity to stimulate splenocyte proliferation. Two wild-growing fruiting bodies were extracted for comparison. Olive oil press cakes reduced the mushroom yield, and the best biological efficiency was obtained on substrates supplemented with wheat bran and without olive oil press cakes. All extracts were capable of inducing splenocyte proliferation and were half as effective as the positive control (6.0 microg/mL phytohaemagglutinin). No correlation between substrate composition and bioactivity could be established. Extracts from wild-growing G. frondosa were superior to cultivated ones in respect to biological activity.