The present study was aimed at evaluating the cytotoxic potential of selected halophilic bacterial metabolites. The use of the metabolomics approach in identifying the unexplored bioactive metabolites from halophilic bacterial isolate reduces time and complex experiments. In our study, we used UV/Visible spectroscopy, LC–MS/MS, and NMR to identify the metabolites present in the methanolic extract of the halophilic bacterium Bacillus VITPS16. MTT assay revealed that metabolite fractions (S1-79.61% and S2-85.74%) possess cytotoxic activity. Colonogenic assay confirmed the cytotoxic potential of the fractions and apoptosis assays showed that 83.37% of the cells undergo apoptosis at 10 mg/mL concentration (MF-S2). The DNA binding studies revealed the metabolite fraction interacts with DNA resulting in cytotoxicity. The study states that MF- S2 induced an antiproliferative effect that led to apoptosis through DNA binding as one of the possible pathways. The toxicity analysis using zebrafish indicated that the metabolite fractions are non-toxic even at 10 mg/mL concentration. Fraction MF-S2 is found to contain phosphoethanolamines, glycerophospholipids, sphingolipids, apocarotenoid, enigmol and its analogue, ankaflavin and flavonoid type of metabolites, which have been previously reported to have anti-cancer activity. © 2021, King Abdulaziz City for Science and Technology.