From an extract of the Okinawan sponge Petrosia strongylata, five novel acetylenic metabolites, petrosiols A (1)–E (5), were isolated and chemically characterized. These compounds induced nerve growth factor (NGF)-like neuronal differentiation of PC12 cells. The structure–activity relationships of the petrosiols, their derivatives, and some related known compounds suggest the importance of the rare 2,4-diyne-1,6,7,8-tetraol fragment for the neurotrophic activity of the petrosiols.