Arboviruses transmitted by Aedes aegypti, such as dengue, Zika, and chikungunya, pose significant public health threats. The biological agent Bacillus thuringiensis (Bt) has been explored as an effective, safe, and economically sustainable method to reduce mosquito populations. This study aimed to identify Bt strains with potent larvicidal activity against Ae. aegypti, contributing to the development of effective bioinsecticides. We evaluated 13 Bt strains from the Embrapa Maize and Sorghum collection against Ae. aegypti larvae. Five replicates of each strain were prepared in 200 mL containers, each containing 18 mL of autoclaved distilled water, 2 mL of bacterial suspension (10⁸ spores/mL), and 20 third-instar Ae. aegypti larvae. Mortality rates were monitored by counting live and dead larvae at 1, 24, 48 and 72 hours. PCR characterization was used to identify genes associated with toxicity for lepidopteran and dipteran pests in three strains. The bioassays confirmed the toxicity of the larvae, with strains 1644 and 1608A causing 100% and 95% mortality, respectively, within 24 hours. Strain 1656 showed 85% mortality up to 72 hours. Larvae exposed to Bt suspensions of strains 1644, 1608A, and 1656 lost agility, their tegument lost their shine, and the color became opaque and eventually darkened after death. Characterization of strain 1656 revealed the presence of cry and vip genes, which are associated with toxic activity in dipterans. The high toxicity of Bt strains 1644, 1608A, and 1656 underscores their potential as biocontrol agents against Ae. aegypti larvae.