To explore the mechanism involved in the inhibition of biofilm formation and antibacterial action of endophytic Streptomyces sp. isolated from the Indian Sundarbans Mangrove Forest. In this study, Streptomyces sp. KJ7TB2 was isolated from the Indian Sundarbans. An ethyl acetate extract was subjected for GC-MS profiling. The mechanisms of antibacterial and antibiofilm action were also explored. In-silico molecular docking and dynamics simulation analyses were performed. The GC-MS analysis of the extract revealed 14 distinct bioactive secondary metabolites. The extract exhibited potent MIC values of 250 μg/mL and 125 μg/mL against Escherichia coli MTCC 1195 and Pseudomonas aeruginosa ATCC 27853. Antibiofilm investigation further revealed inhibition of biofilm formation in both pathogens especially in E. coli MTCC 1195, likely by disrupting initial surface adhesion and exopolysaccharide synthesis. Also, the extract inhibited swarming motility, suggesting interference with flagella-driven motility and quorum sensing. The brine shrimp lethality assay confirmed a favorable safety profile, recording limited mortality (36.84% at 2×MIC over 24 h). In-silico molecular docking and dynamics simulation analyses exhibited robust affinity for two key biofilm-associated proteins, exhibiting significant binding energy (ΔG). The study highlights Streptomyces sp. KJ7TB2 as a potential source for the discovery of safe, potent therapeutics.