The studied compound SN12 significantly inhibited biofilm formation in Pseudomonas aeruginosa, but its regulatory mechanism is unknown. To determine the mechanism of action, TMT tagging was used to analyse the proteomics of P. aeruginosa PAO1. Three groups of blank controls treated with PBS were compared with three groups of experimental groups treated with 1 μM compound to assess its effect. Protein expression was identified and analysed using RPLC-MS technology subsequent to the extraction of the entire proteome.