Water-filled tree holes are important temporal ecosystem for a number of different aquatic and semi-aquatic organisms. The study of tree-hole breeding mosquitoes was carried out in the Annamalai University campus, Tamilnadu, India between June 2013 - February 2014, by using standard entomological procedures. The tree holes are sampled for various mosquito species around the campus. Types of mosquito species encountered, their relative abundance, as well as genera varied significantly during the study. Three genera of mosquitoes: Aedes, Culex, Anopheles were recovered while 14 species of mosquitoes encountered include: Aedes aegypti, Ae.albopictus, Ae.africanus, Ae.simpsoni, Ae.taylori, Culex quinquefaciatus, Cx.nebulosus, Cx.tritaeniorhynchus, Cx.pseudovishnui, Cx.pipens, Anopheles stephensi, An.sundaicus, An.culiciformis and An.fluviatilis. Most of the mosquitoes showed preference to tree holes for their oviposition. Tree hole mosquitoes are studied by α-biodiversity like Margalef index (DMg), Simpson’s index (λ), Shannon-Wiener (H’), Pielou’s index (J’). The presence of Ae.aegypti and Ae.albopictus indicate that the study areas were at risk of dengue fever epidemic. The presence of Anopheles and Culex species ensured endemicity of malaria and filariasis, while the recovery of Ae.aegypti in this region suggests a possible outbreak of dengue fever in future if not properly controlled.