Polymicrobial biofilm can form on implants or devitalized tissue, leading to severe musculoskeletal infections. Polymicrobial infection is associated with lower treatment success rates, longer hospitalization times, increased numbers of surgical procedures, and amputations. The presence of both gram-positive and gram-negative microorganisms adds to treatment difficulty and cost because either broad-spectrum or multiple antimicrobials are required for successful treatment. In vitro and ex vivo coculture models are valuable in initial evaluations of antimicrobial combination devices but do not fully recapitulate the infection microenvironment. In vivo models of infection established using single microorganism contaminants require further refinement to model polymicrobial infection treatment outcomes. In this study, we adapted an established murine orthopedic model combining Staphylococcus aureus and Pseudomonas aeruginosa in a polymicrobial biofilm-associated infection model. In a preliminary study to establish the model, Pseudomonas presence in the bone was increased in the presence of S. aureus, although Pseudomonas did not form biofilm on a stainless-steel Kirschner wire in the defect. The colonies of S. aureus retrieved predominate in the bone at up to 100-fold greater numbers, although S. aureus biofilm formation was limited compared to monomicrobial models. Although increasing the inoculum of Pseudomonas did not affect the staphylococcal biofilm formation on the wire, there were trends toward increased average S. aureus bone colonization as P. aeruginosa concentration in the inoculum increased. Further model validation will consider the inoculation site(s), isolation points from clinical microbiological strains, epidemiology of clinical polymicrobial communities, ratio of coinoculation, effects of biofilm, and outcome measures. Murine models of polymicrobial infection will be a valuable tool for assessing the feasibility and efficacy of antimicrobial combination devices in the prevention or treatment of infection.