The mosquito Aedes aegypti vectors arboviruses such as urban yellow fever, dengue, chikungunya and Zika. Reducing the vector population is still the most effective method to decrease the spread of arboviruses. Fungi are the main natural disease agents of insects. The present study compared the virulence of conidia and blastospores of the entomopathogenic fungus Metarhizium anisopliae against A. aegypti pupae. The pupae were obtained by collecting eggs using “ovitraps” deployed in an urban environment. M. anisopliae conidia were produced using solid media and blastospores were produced by inoculating conidia in liquid culture. Blastospores were more virulent against pupae than conidia, and no pupae survived twenty-four hours after exposure to this type of propagule. Large quantities of mucilage were produced by the blastospores in the presence of the pupae. Exposure of pupae to conidial suspensions resulted in 57% survival at 24h and 23% at 48h. A proportion of the adults, which emerged from pupae exposed to conidia, succumbed to infection. This is the first study to demonstrates the infection of A. aegypti pupae by the entomopathogenic fungus M. anisopliae, showing that this stage of development can also be targeted by biological control agents.