Microbial biosurfactant has wide applications in industrial sectors owing to their eco-friendly and biocompatible nature giving, an alternative to chemically synthesized surfactants. In our study, Kocuria marina DAG II, an isolated bacterium was observed to produce a biosurfactant. The production of biosurfactant was established by oil-spreading (3.72 ± 0.15 cm), drop collapse, and CTAB (Cetyltrimethyl ammonium bromide) assay. Biosurfactant activity was re-confirmed by the reduction in surface tension of uninoculated broth from 68.52 mN/m to 47.40 ± 0.57 mN/m. Chemical characterization study of the biosurfactant was done by TLC (thin-layer chromatography) indicating the presence of proteins (Rf = 0.78 ± 0.08), lipids (Rf = 0.79 ± 0.09), and polysaccharides (Rf = 0.81 ± 0.06). FTIR and NMR (1HNMR and 13CNMR) studies confirmed the presence of different functional groups and revalidated the presence of amino acids, lipids, and polysaccharides in the biosurfactant. From the optimization study, the highest production of biosurfactant having a yield of 3.05 ± 0.05 g/L was obtained at a temperature of 31 °C and pH 8, when kept for an incubation period of 88 hr. The addition of rancid and waste-frying mustard oil and used engine oil could enhance the production of biosurfactant by Kocuria marinaDAGII hence substantiating the concept of value-added biosurfactant production from waste. Kinetic modeling showed a good fit between experimental and predicted values using modified Gompertz model with R2 values of 0.99 and 0.99. The biosurfactant exhibited radical scavenging activity through DPPHand ABTS assays with IC50 values of 1.29 mg/ml and 0.56 mg/ml respectively. The biosurfactant produced by Kocuria marina DAG II could be utilized for various applications, bypassing the need for a synthetic surfactant.