Escherichia coli is a major concern in the food industry because of its ability to produce biofilm causing food contamination. Autoinducer 2 (AI-2) in E. coli is used to regulate quorum sensing (QS) which controls its physiological functions and pathogenicity, especially biofilm formation. Inhibition of the QS is an alternative decontamination approach in the food industry. In this study, 9 bacterial strains isolated from mangrove forests demonstrated their ability to inhibit the bioluminescent production of Vibrio campbellii biosensor strain. Bacteria were identified and Staphylococcus warneri was chosen for further characterization. The minimum inhibitory concentration (MIC) of the cell-free supernatant (CFS) extract against E. coli ATCC 25922 was 25 mg/mL. The CFS extract at 1/2- and 1/4-MIC showed 70–100% and 28–62% biofilm reduction against biofilm-forming E. coli, respectively. The reduction of biofilm and cell attachment was observed under SEM. LC/MS analysis demonstrated that 21 compounds were identified and the active compound on the QS will further be investigated in future research. From our study, we concluded that S. warneri isolated from mangrove forests can produce bioactive compounds interfering QS system, inhibiting E. coli biofilm and demonstrating the potential to develop a novel biocontrol agent for decontamination of biofilm-forming bacteria contaminated in food production.