Significance In laboratory experiments, Wolbachia ( w Mel strain)-infected Aedes aegypti are refractory to disseminated arboviral infections. Yet previous characterizations of w Mel-mediated blocking have not considered several biologically and ecologically important factors likely to influence the virus–mosquito interaction. After direct feeding on 141 viremic dengue patients, we demonstrate w Mel lowers dengue virus (DENV) transmission potential and lengthens the extrinsic incubation period. Subsequently, using established field populations of wild-type and w Mel-infected Ae. aegypti , we compared field- versus laboratory-rearing conditions on mosquito susceptibility to disseminated DENV infection. The magnitude of w Mel-mediated virus blocking was even greater when mosquitoes developed under field conditions. These clinically and ecologically relevant findings support Wolbachia introgression into Ae. aegypti populations as a biocontrol method to reduce the transmission of DENV and other arboviruses.