Effects of ampicillin (Amp) in combination with riboflavin on septic arthritis in mice infected with Staphylococcus aureus have been reported. Ampicillin was given at 100 mg/kg after 24 h of infection, followed by riboflavin (Ribo) at 20 mg/kg body wt, after 2 h of Amp treatment. Mice were sacrificed at 3, 9, 15 days post infection (dpi). Combined treatment of infected mice with ampicillin and riboflavin eradicated the bacteria from blood, spleen and synovial tissue and showed a significant gross reduction in arthritis, reduced serum levels of TNF-α and IFN-γ. S. aureus infected mice exhibited higher synovial TNF-α and IL-6, which was also reduced by ampicillin and riboflavin treatment. S. aureus infected mice showed a disturbed antioxidant status measured in terms of cellular anti-oxidants like reduced glutathione and anti-oxidant enzymes such as superoxide dismutase and catalase and were ameliorated when the animals were co-treated with ampicillin along with riboflavin. Results of the study showed that combined treatment with anti-oxidant and antibiotic may protect from staphylococcal arthritis and may ameliorate oxidative stress caused by S. aureus infection.