Background & objectives: Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated. Methods: Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2nd – 3rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA. Results: There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2nd, 3rd and 4th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested. Interpretation & conclusion: The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.