ABSTRACT A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [ ¹⁸ F]fluoro-deoxyglucose–MicroPET ([ ¹⁸ F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [ ¹⁸ F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [ ¹⁸ F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches.