Biosurfactants are amphiphilic surface-active molecules of microbial origin principally produced by hydrocarbon-degrading bacteria; in addition to the bioremediation properties, they can also present antimicrobial activity. The present study highlights the chemical characterization and the antimicrobial activities of biosurfactants produced by deep-sea marine bacteria from the genera Halomonas, Bacillus, Streptomyces, and Pseudomonas. The biosurfactants were extracted and chemically characterized through Chromatography TLC, FT-IR, LC/ESI-MS/MS, and a metabolic analysis was done through molecular networking. Six biosurfactants were identified by dereplication tools from GNPS and some surfactin isoforms were identified by molecular networking. The half-maximal inhibitory concentration (IC ) of biosurfactant from Halomonas sp. INV PRT125 (7.27 mg L ) and Halomonas sp. INV PRT124 (8.92 mg L ) were most effective against the pathogenic yeast Candida albicans ATCC 10231. For Methicillin-resistant Staphylococcus aureus ATCC 43300, the biosurfactant from Bacillus sp. INV FIR48 was the most effective with IC values of 25.65 mg L and 21.54 mg L for C. albicans, without hemolytic effect (< 1%), and non-ecotoxic effect in brine shrimp larvae (Artemia franciscana), with values under 150 mg L , being a biosurfactant promising for further study. The extreme environments as deep-sea can be an important source for the isolation of new biosurfactants-producing microorganisms with environmental and pharmaceutical use.