Osteomyelitis is a well-known bone infection in humans. The primary symptoms are fever, pain, weakness, and redness of the bone. l-Homocarnosine is a bioactive peptide abundant in brain and skeletal muscles. The present study evaluated the synergistic effect of vancomycin and l-homocarnosine against osteomyelitis in the Staphylococcus aureus-induced rat model of osteomyelitis. Animals were classified into the following groups: sham (group I), osteomyelitis (group II, control), vancomycin (25 mg/kg body weight, group III), l-homobrassinolide (25 mg/kg body weight, group IV), and vancomycin (25 mg/kg body weight) + l-homobrassinolide (25 mg/kg body weight) (group V). Lipid peroxidation, superoxide dismutase (SOD), glutathione peroxidase (Gpx), catalase, reduced glutathione (GSH), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined. Assessments of bacterial growth and histopathological analyses were carried out. Lipid peroxidation, GSH, SOD, catalase, and Gpx levels recovered to near normal levels with the combined treatment of vancomycin and l-homocarnosine. TNF-α and IL-6 levels were reduced to near normal levels. Combined supplementation of vancomycin and l-homocarnosine reduced bacterial growth in bone and wire. Furthermore, bone infections and histopathological scores were also reduced. In summary, we showed that combined treatment of vancomycin and l-homocarnosine was more effective against bacterial growth and bone infection compared to monotherapy with vancomycin or l-homocarnosine.