Pseudomonas aeruginosa is an important etiological agent associated with systemic infection in burn patients. Quorum sensing (QS) mechanism of P. aeruginosa contributes to its virulence. Inhibition of QS signals can serve as an effective anti-virulence strategy. Aim of the present study was to evaluate the efficacy of lactonase alone and in combination with ciprofloxacin in treating P. aeruginosa murine burn wound infection. Topical application of lactonase alone and ciprofloxacin alone prevented systemic spread of P. aeruginosa through burned skin and also reduced the mortality. When lactonase (topical) and ciprofloxacin (I/P) were used in combination, zero mortality was observed. It not only significantly reduced systemic dissemination and severity of histopathologic lesions but also resulted in skin regeneration. Decreased production of pathologic index factors (malondialdehyde and reactive nitrogen intermediates) and interleukins (IL-10, IL-6 and MIP-2) was also observed in comparison to control. The results of present study suggest that combination of lactonase and ciprofloxacin can potentially attenuate the virulence of P. aeruginosa. This is the first report of topical administration of lactonase along with antibiotic for the efficient control of burn wound infection.