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Extracellular polymeric substances (EPS) are complex, hydrated matrices produced by biofilm-forming bacteria to anchor themselves to surfaces and resist antibiotic treatment. EPS plays a critical role in the formation, maintenance and virulence of biofilms, leading to persistent infections and posing significant challenges in healthcare. Characterizing bacterial EPS is essential to understand their biochemical composition and functional properties, which is critical for optimizing their applications in biotechnology, medicine, and environmental management. Therefore, the present study aimed to isolate and screen the bacteria from Arabian Sea for their ability to produce EPS from biofilm surfaces. Additionally, the detailed characterization of the EPS was also carried out. These bacteria were identified using 16S rRNA gene sequence analysis and revealed that all the EPS-producing bacterial isolates belong to different bacterial genera (Oceanimonas, Psychrobacter and Vibrio). The bacteria were cultured on Zobell marine broth media and EPS were isolated using the propanol precipitation method. The EPS weight varied among the bacterial isolates and ranged from 0.81 g L−1 to 2.21 g L−1. The EPS produced by the bacterial strains have shown antimicrobial as well as free radicals (2,2-diphenyl-1-picrylhydrazyl; DPPH) and 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity. The antimicrobial and free radical scavenging properties of EPS have promising biotechnological applications in developing new antibiotics, enhancing food preservation, creating protective coatings, and improving wound healing therapies. The Fourier-transform infrared spectroscopy (FTIR) revealed the presence of aliphatic methyl, halide groups, saccharides and primary amines. Gas chromatography equipped with mass spectroscopy (GC–MS) confirmed the presence of monosaccharides such as glucose, galactose, arabinose and mannose. The EPS were further characterized using X-Ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The study highlights the importance of EPS in biofilm formation, antibiotic resistance and persistent infections, emphasizing the importance of isolating and characterizing of EPS for its potential biotechnological applications, including antimicrobial and free radical scavenging activities. [ABSTRACT FROM AUTHOR]