Abstract Marine sponges represent an affluent source of biogenetically unprecedented array of biologically active compounds. This study revealed the isolation of ten compounds from marine sponge of Petrosia sp. Their chemical structures were determined by using 1D and 2D NMR, UV, IR and MS measurements. A polyoxygenated steroid (3β,7β,9α-trihydroxycholest-5-en (1), a purine-derivative (3,7-dimethyl-2-(methylamino)-3H-purin-6(7H)-one (2) and a sphingolipid (N-((3S,E)-1,3-dihydroxytetracos-4-en-2-yl)stearamide (3) proved to be new compounds. Meanwhile, seven known compounds; (4–10) were also identified. The cytotoxicity of the total extract and the isolated compounds were subjected to cytotoxicity evaluation employing two cancer cell lines; HepG2 and MCF-7. All tested compounds exhibited cytotoxic effect on both cancer cell lines with IC50 in range of 13–98μg/mL. The proposed mechanism of cytotoxic activities was examined through its molecular affinity to the DNA. Compound 5 showed the highest affinity to the DNA with IC50 30μg/mL.