Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets , a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of in the in vivo filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify elimination in filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in , , and in vivo preclinical models of filarial disease and in vitro selectivity against (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.