Significance In laboratory experiments, Wolbachia (wMel strain)-infected Aedes aegypti are refractory to disseminated arboviral infections. Yet previous characterizations of wMel-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 wMel lowers dengue virus (DENV) transmission potential and lengthens the extrinsic incubation period. Subsequently, using established field populations of wild-type and wMel-infected Ae. aegypti, we compared field- versus laboratory-rearing conditions on mosquito susceptibility to disseminated DENV infection. The magnitude of wMel-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. The wMel strain of Wolbachia can reduce the permissiveness of Aedes aegypti mosquitoes to disseminated arboviral infections. Here, we report that wMel-infected Ae. aegypti (Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. The wMel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing of wMel introgression for the biocontrol of Ae. aegypti-born arboviruses.