Background <p>Constant research is being conducted to discover potent antibacterial and anticancer agents from natural sources like plants and microorganisms. Gram-positive <i>Paenibacillus dendritiformis</i> is a spore-forming bacterium with recognized industrial and economic significance in the production of important biomolecules applicable to the agricultural, pharmaceutical, and biotechnological industries.</p> Objectives <p>The objective of this study was to investigate the antibacterial and cytotoxic potential of bioactives produced by <i>P. dendritiformis</i> isolated from Lonar Lake sediment.</p> Methods <p><i>P. dendritiformis</i> was isolated from Lonar Soda Lake and characterized using colony morphology, biochemical, and phylogenetic evaluations. The process was carried out using starch casein broth (SCB), and the bioactives were characterized by LC–MS/MS. The antibacterial assay was performed by the disc diffusion assay. The cytotoxic activity was performed by employing HeLa cell culture. To understand the molecular mechanism, oxidative stress markers, pro-inflammatory markers, and apoptotic markers were evaluated.</p> Results <p>LC–MS/MS spectra reveal the presence of phytyl palmitate and phytol acetate. These bioactives from <i>P. dendritiformis</i> exhibit moderately significant to significant antibacterial and cytotoxic activity by substantially inhibiting the HeLa cell viability via MTT assay.</p> Conclusion <p>The study demonstrates the antibacterial and cytotoxic potential of bioactive compounds from <i>P. dendritiformis</i> with significantly lower IC₅₀ values as compared to standard probes.</p> Graphical Abstract <p></p>