The inadequacy of standard mosquito control strategies calls for ecologically safe novel approaches, for example the use of biological agents such as the endosymbiotic α‐proteobacteria Wolbachia or insect‐specific viruses (ISVs). Understanding the ecological interactions between these “biocontrol endosymbionts” is thus a fundamental step. Wolbachia are transmitted vertically from mother to offspring and modify their hosts’ phenotypes, including reproduction (e.g., cytoplasmic incompatibility) and survival (e.g., viral interference). In nature, Culex pipiens (sensu lato) mosquitoes are always found infected with genetically diverse Wolbachia called wPip that belong to five phylogenetic groups. In recent years, ISVs have also been discovered in these mosquito species, although their interactions with Wolbachia in nature are unknown. Here, we studied the interactions between a widely prevalent ISV, the Culex pipiens densovirus (CpDV, Densovirinae), and Wolbachia in northern Tunisian C. pipiens populations. We showed an influence of different Wolbachia groups on CpDV prevalence and a general positive correlation between Wolbachia and CpDV loads. By investigating the putative relationship between CpDV diversification and wPip groups in the different sites, we detected a signal linked to wPip groups in CpDV phylogeny in sites where all larvae were infected by the same wPip group. However, no such signal was detected where the wPip groups coexisted, suggesting CpDV horizontal transfer between hosts. Overall, our results provide good evidence for an ecological influence of Wolbachia on an ISV, CpDV, in natural populations and highlight the importance of integrating Wolbachia in our understanding of ISV ecology in nature.