Wolbachia mediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect, Wolbachia and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, indicating that oxidative stress may be important for Wolbachia-mediated antiviral protection. However, Wolbachia experimentally introduced into mosquitoes impacts a range of host fitness traits some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection in Wolbachia-infected insects we analysed oxidative stress of five Wolbachia-infected Drosophila lines. In flies infected with protective Wolbachia strains hydrogen peroxide concentrations were 1.25- to 2-fold higher than in paired fly lines cured of Wolbachia infection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with non-protective Wolbachia strains compared to flies cured of Wolbachia infection. Using a Drosophila mutant that produces increased levels of hydrogen peroxide we investigated whether flies with high endogenous reactive oxygen species had altered response to virus infection and found flies with high endogenous levels of hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates with Wolbachia-mediated antiviral protection in natural Drosophila hosts.