Chronic wound infections are caused by biofilm forming opportunistic pathogenic bacteria. The persistence of infection, co-infecting pathogens and prolonged use of antibiotics promote antibiotic resistance hampering healing process due to increased inflammation. Hence, we tested the broad range antibacterial activity of linalool, a bioactive monoterpene commonly present in many essential oils having anti-inflammatory and antimicrobial activities to target different opportunistic pathogens commonly found in the chronic wound. We included some of the common pathogens such as Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus, to study the broad range antimicrobial efficacy of linalool. The in vitro effect of linalool on biofilm was quantified in pre-treatment, post-treatment, repetitive treatment, and polymicrobial biofilm scenarios. Time-kill and XTT (2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2 H-tetrazolium-5-carboxanilide) assays were performed to confirm the efficacy of linalool against wound infections, and these results were further validated using simulated wound exudates medium (WEM) which mimics the wound environment. The mechanism of bactericidal action was determined using assays for membrane integrity and oxidative stress. The results indicated the broad range antimicrobial activity of linalool with minimum inhibitory concentration (MIC) ranging from 2.5 to 5 µL/mL against E. coli, A. baumannii, E. faecalis, S. aureus, and K. pneumoniae, while for P. aeruginosa the MIC was 20 µL/mL. Linalool was most effective against E. coli, E. faecalis, K. pneumoniae, A. baumannii, and S. aureus, and could inhibit the growth and biofilm by more than 90% and 80%, respectively, at 5 µL/mL. The XTT assay confirmed the MIC results, showing a significant reduction in the metabolic activity of the pathogens (p <
0.001). In the simulated WEM similar response of the bacteria to linalool treatment was observed. At 5 to 20 µL/mL concentrations, linalool significantly inhibited the polymicrobial biofilm consisting of P. aeruginosa, A. baumannii, and S. aureus in two species combinations. The mechanism of bactericidal action was associated with the increased reactive oxygen species production and disruption in the membrane integrity leading to release of cellular content. The anti-inflammatory activity of linalool, assessed using the albumin denaturation method showed significant activity at the tested concentrations. In conclusion, the findings suggest the therapeutic potential of linalool in treating biofilm associated chronic wound infections due to its versatile broad spectrum activity.