Treatment of infected burn wounds with topical antibiotics is challenging, particularly those invaded with methicillin-resistant Staphylococcus aureus bacteria (MRSA). This is due to the formation of bacterial biofilms characterized by antimicrobial resistance. Mupirocin (MP), a widely used topical antibiotic, is active against gram-positive bacteria including MRSA. However, MP suffers from sub optimal therapeutic efficacy due to its poor water-solubility and the significant rise in MP-resistant S. aureus. In this study, the physico-chemical characteristics of MP were modified through nanocrystallization to improve its therapeutic efficacy for the treatment of skin infections. Mupirocin nanocrystals (MP-NC) were prepared using a nanoprecipitation technique and optimized using a D optimal response surface design. The optimization of MP-NC produced ultra-small monodisperse spherical particles with a mean diameter of 70 nm and a polydispersity index of 0.2. The design resulted in two optimal MP-NC formulations that were evaluated by performing series of in vitro, ex vivo, microbiological, and in vivo studies. In vitro results showed a 10-fold increase in the saturation solubility and a 9-fold increase in the dissolution rate of MP-NC. Ex vivo permeation studies, using pig ears skin, showed a 2-fold increase in the dermal deposition of MP-NC with the highest drug deposition occurring at 500-µm skin depth. Moreover, the optimal MP-NC formulations were lyophilized and incorporated into a 2% w/w cream. Microbiological studies revealed a 16-fold decrease in the minimum inhibitory concentration and the minimum bactericidal concentration of MP NC. In vivo studies, using a rat excision burn wound model, demonstrated rapid and complete healing of infected burn wounds in rats treated with MP-NC cream in comparison to marketed Mupirocin ointment (Avoban ointment 2% w/w, Avalon Pharma). Our results suggest that nanocrystallization of MP may provide an avenue through which higher levels of a topically appli