Weeds in rice fields compete with crops for essential resources, causing severe yield losses based on weed infestation levels and control measures. The current study, conducted at Mahatma Gandhi University, Kottayam, Kerala, India, during June 2023 and March 2024, aims to assess the potentiality of Kluyvera intermedia as a bacterial biocontrol agent against Echinochloa crus-galli (L.) P. Beauv. (barnyard grass), the common weed of rice fields. Of the 127 bacterial isolates, the bacterial strain Kluyvera intermedia MA2 efficiently controlled Echinochloa crus-galli. The treated plants showed chlorosis after the first 3 days of application of bacteria and complete death of the plant in 7 days. The study’s second objective was to identify the active compound responsible for the herbicidal activity of Kluyvera intermedia. The characterization of the active compounds via HR LC-MS/MS (Q-TOF) analysis revealed phthalic acid esters, pyrazine derivatives, and quinoline derivatives as bioactive compounds. Molecular docking studies with antioxidant enzymes revealed significant interactions between phthalic acid esters and key amino acid residues: SER173, ARG38, and ALA134 of ascorbate peroxidase; VAL372 and SER374 of glutathione reductase. Propylpyrazine showed strong binding with PRO367 of catalase, ILE93 of glutathione S-transferase, ARG31 of ascorbate peroxidase, and ASP466 of glutathione reductase. Additionally, 6-methylquinoline interacted notably with ALA253 of catalase. Biochemical, enzymatic, and antibiotic profiling identified the bacterium as an IAA-producing, gram-negative and rod-shaped strain. It demonstrated susceptibility to eight antibiotics and the ability to produce several enzymes, including cellulases, catalases, phenylalanine deaminases, and proteases.