Molecular Insights Into Terpinen‐4‐ol's Antimicrobial Action: Targeting SarA and OprF in Bacterial Biofilm Formation

Biofilms, which are protective microbial communities, worsen antimicrobial resistance by shielding pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa from treatment. This research explored the effectiveness of tea tree oil (TTO) ( Melaleuca alternifolia ) and its main component, terpinen‐4‐ol, against these tough structures. Chemical analysis using GC‐MS showed that terpinen‐4‐ol made up 45.23% of the M. alternifolia oil composition. In vitro tests revealed strong antibacterial properties, with a minimum inhibitory concentration (MIC) of 0.39% for both pathogens. Confocal microscopy demonstrated that concentrations below the MIC effectively disrupted the 3D biofilm architectural analysis and reduced thick biomass. To uncover the mechanism behind this, in silico molecular docking and 100 ns GROMACS simulations were conducted. Terpinen‐4‐ol targeted the SarA regulator (2FNP) in S. aureus and the OprF protein (4RLC) in P. aeruginosa , forming stable complexes through hydrophobic interactions and hydrogen bonds. Although toxicity profiling indicated potential skin sensitization, it predicted low acute toxicity. These results suggest that terpinen‐4‐ol is a potent anti‐virulence agent capable of reducing persistent contamination in clinical and food‐processing settings.