As part of our ongoing studies on bioactive natural products from marine sponges, we investigated the cytotoxic potential of extracts from the new sponge Petrosia tuberosa sampled from Mauritius waters. Bioguided fractionation of the ethyl acetate extract by vacuum liquid chromatography (VLC) revealed two fractions, namely VLC (6-9) and (13-17) showing cell deaths of 86 ± 1% and 88 ± 4%, respectively, at 50 μg/mL on HeLa cells. At 10 μg/mL, only VLC (13-17) displayed a significant cell death (56 ± 7%) compared with VLC (6-9) (8 ± 1%). The cytotoxic activity of VLC (13-17) was also determined on nine other human cancer cell lines. Clonogenic assay, mitochondrial membrane potential change, DNA fragmentation and microscopic analysis of fraction VLC (13-17) revealed distinct features of apoptosis on HeLa cells. Further fractionation and purification of this fraction by chromatographic techniques resulted in isolation of one known secondary metabolite, petrosynol. Its structure was determined by 1H and 13C-NMR analyses.