Microbial flocculants are versatile class of novel biomacromolecules with numerous potential industrial applications. This study sought to investigate the antimicrobial, antioxidant, and anti-inflammatory potential of a polysaccharide-based bioflocculant (PBB) extracted from Bacillus subtilis F9. To achieve this, the antioxidant activity of different PBB concentrations(100 μg/mL ̶ 1000 μg/mL) was first examined in vitro using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radicals, and superoxide radical scavenging assays. Further, the anti-inflammatory activity of PBB against lipopolysaccharide (LPS; 1 μg/mL)-induced inflammatory mediators released from headkidney (HK)-derived macrophages of Labeo rohita was investigated. Our results revealed that the capacities of 800 μg/mL of PBB to scavenge DPPH, hydroxyl radicals, and superoxide radicals were 81.46 ± 1.37%, 66.34 ± 2.63%, and 78.03 ± 2.46%, respectively, which were slightly higher that observed following treatment with 400 μg/mL of the positive control (ascorbic acid). Further, the radical scavenging capacity of PBB was found to steadily increase with increasing concentrations of PBB. Pre-treatment with PBB also inhibited nitric oxide production in a dose-dependent manner. We next examined the effect of PBB on proinflammatory cytokines (TNF-α, and IL-1β) and anti-inflammatory cytokines (IL-10, TGF-β) via qRT-PCR and ELISA. We found that PBB markedly inhibited the LPS-induced mRNA and protein expression levels of TNF-α and IL-1β, while it significantly increased those of IL-10 and TGF-β. Further, PBB exhibited an antibacterial activity against multiple food-borne pathogens with minimal inhibitory concentration values in the range of 3 ̶ 11 mg/mL. Importantly, PBB exhibited negligible cytotoxic effects against HK macrophages. Taken together these results suggest that PBB may serve as a natural antioxidant for application in functional therapies and may also be exploited for its anti-inflammatory potential.