Mosquito-borne diseases such as dengue and West Nile virus pose serious public health risks. On Madeira Island, the presence of the mosquito species Aedes aegypti (Linnaeus, 1762) and Culex pipiens (Linnaeus, 1758) raises concerns about local transmission. In this study, we tested 100 Ae. aegypti and 40 Cx. pipiens mosquitoes collected exclusively in the municipality of Funchal, Madeira Island, to assess the presence and diversity of Wolbachia, a naturally occurring bacterium known to reduce mosquitos' ability to transmit viruses. Molecular identification confirmed that all Cx. pipiens specimens belonged to the molestus biotype, with three individuals identified as hybrids between molestus and pipiens biotypes. This is the first evidence of such hybrids in Madeira. Wolbachia was not detected in any of the Ae. aegypti samples. In contrast, all Cx. pipiens mosquitoes were positive, showing a 100% prevalence. Genetic characterization placed these infections within the wPip clade, supergroup B, sequence type 9. These findings provide key baseline data to inform future mosquito control strategies on the island. As Ae. aegypti showed no natural Wolbachia infection, introducing Wolbachia-infected mosquitoes may be necessary to implement such biocontrol approaches in Madeira.