Biofouling leads to water quality deterioration and higher maintenance cost for cleaning of membranes. The present study has demonstrated the application of a biomolecule (vanillin) in scrubbing and destabilizing biofilms of drinking water reverse osmosis (RO) membrane module in lab scale reactor set-up. Reverse osmosis membrane reactor was connected with tap water supply and subjected with optimal concentration of vanillin. The pressure drop was delayed by 17-20 days as compared to control reactor. Real-time PCR analysis of metagenome indicated the reduced copy number of functional biofilm-associated genes (bdlA, lasI, pgaC) in treated membrane. SEM and metagenome analysis revealed that the sticky biofilm communities shifted to loosely bound emboli after vanillin treatment. Metagenome sequence analysis revealed the inhibitory activity against major biofouling biota like members of Proteobacteria, Acidobacteria, Acnitobacteria, Bacteroidetes, Candidatus, Nitrospira, and Firmicutes. Biofouled membrane metagenome sequence was also compared with real-life (brackish water, waste water, domestic drinking water) biofouled membrane communities. In silico docking of vanillin to receptor proteins and chemical configuration simulation along with other phenolic derivatives were performed, which suggested that the autoiducer signal capability of vanillin was effective against representative broad spectrum biofilm population. Vanillin exhibited the quorum-quenching mode of action by virtue of docking towards similar amino acid (Thr 131, Ilu 214) responsible of autoinducer signal anchoring in the transcriptional regulatory proteins.