The unique properties of biosurfactants obtained from microbes, including their activity at extreme temperatures, make them more attractive than synthetic alternatives. Henceforth, the principle objective is to isolate and detect the antibacterial and antifungal activities of the biosurfactants produced from bacteria of the economically competitive mangrove ecosystem. Using the serial dilution method, 53 bacterial strains were recovered from the Manakudy mangrove forest in Kanyakumari, India, for the investigation. Different biosurfactant screening methods and morphological and biochemical tests were opted to select the potential biosurfactant producer. After the initial screening, two strains were discovered by 16S rRNA gene sequencing followed by extraction using chloroform: methanol (2:1) by the precipitation method. The partially purified biosurfactants were then screened for antimicrobial properties against pathogens including Mucor sp., Trichoderma sp. Morphological, biochemical, and 16S rRNA gene sequencing identified the two strains to be gram-positive, rod-shaped bacteria namely Virgibacillus halodentrificans CMST-ZI (GenBank Accession No.: OL336402.1) and Pseudomonas pseudoalcaligenes CMST-ZI (GenBank Accession No (10K): OL308085.1). The two extracted biosurfactants viz., 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester, as well as cycloheptane efficiently inhibited human pathogens, including Enterococcus faecalis, and fungi, including Mucor sp., Trichoderma sp., indicated by the formation of a zone of inhibition in pharmacological screening. Thus, there is a growing interest in the prospective application of these biosurfactants isolated from marine microbes, exhibiting antimicrobial properties which can be further studied as a potential candidate in biomedical studies and eco-friendly novel drug development.