Background: Marine sponges (phylum Porifera) are sessile filter-feeders from the ocean that are becoming the wealthiest sources of pharmacologically active compounds. Objectives: Our objectives are to identify bioactive compounds from marine sponges (Xestospongia exigua, Xestospongia muta, and Iotrochota baculifera) and to determine their antibacterial activity. Materials and Methods: Methanolic crude extracts were subjected to two-steps fractionation: first, solvent partitioning was conducted using diethyl ether and butanol, followed by column chromatography. The resulting fractions were tested for antibacterial activity against four bacterial strains (Staphylococcus aureus ATCC 25923, Micrococcus luteus ATCC 4698, Escherichia coli ATCC 11775, and Salmonella typhimurium ATCC 14128). The fractions were subsequently profiled using High-Performance Thin Layer Chromatography (HPTLC), and the component of active sub-fractions (SF) was identified using Gas Chromatography-Mass Spectrometry (GC-MS). Results: Although no antibacterial activity was recorded of the methanolic extracts in all marine sponges samples, the response towards diethyl ether extracts of X. exigua was strong. Out of 17 sub-fractions of diethyl ether profiled, three sub-fractions, i.e. 5, 13, and 14 were active. GC-MS identified five compounds in SF 5, four compounds in SF 13, and three compounds in SF 14. Furthermore, SF 13 and SF 14 could inhibit the growth of all bacteria tested, indicating a broad-spectrum activity. On the contrary, SF 5 showed selective inhibition only to E. coli and S. typhimurium, indicating narrow-spectrum activity. Conclusion: Bioactive SF 13 of X. exigua has a high potential as an antibacterial agent but in vitro assessment such as cytotoxicity against mammalian cell lines is needed to determine the toxicity and drug response.