Background Insecticide-treated net (ITN) durability is evaluated using longitudinal bioefficacy and fabric integrity sampling post-distribution. Interceptor® G2 was developed for resistance management and contains two adulticides: alpha-cypermethrin and chlorfenapyr; it is a pro-insecticide that is metabolized into its active form by mosquito-detoxifying enzymes and may be enhanced when the mosquito is physiologically active. To elucidate the impact of bioassay modality, mosquito exposures of the alphacypermethrin ITN Interceptor® and dual adulticide Interceptor® G2 were investigated. Methods This study evaluated the performance of Interceptor® G2 compared to Interceptor® against local strains of mosquitoes in Tanzania. Unwashed and 20× times washed nets were tested. Efficacy of ITNs was measured by four bioassay types: (1) World Health Organisation (WHO) cone test (cone), (2) WHO tunnel test (tunnel), (3) Ifakara ambient chamber test (I-ACT) and (4) the WHO gold standard experimental hut test (hut). Hut tests were conducted against free-flying wild pyrethroid metabolically resistant Anopheles arabiensis and Culex quinquefasciatus. Cone, tunnel and I-ACT bioassays used laboratory-reared metabolically resistant An. arabiensis and Cx. quinquefasciatus and pyrethroid susceptible Anopheles gambiae sensu stricto and Aedes aegypti. Results Against resistant strains, superiority of Interceptor® G2 over Interceptor® was observed in all “free-flying bioassays”. In cone tests (which restrict mosquito flight), superiority of Interceptor® over Interceptor® G2 was recorded. Mortality of unwashed Interceptor® G2 among An. arabiensis was lowest in hut tests at 42.9% (95% CI: 37.3–48.5), although this increased to 66.7% (95% CI: 47.1–86.3) by blocking hut exit traps so mosquitoes presumably increased frequencies of contact with ITNs. Higher odds of mortality were consistently observed in Interceptor® G2 compared to Interceptor® in “free-flying” bioassays using An. arabiensis: tunnel (OR = 1.42 [95% CI:1.19–1.70], p < 0.001), I-ACT (OR = 1.61 [95% CI: 1.05–2.49], p = 0.031) and hut (OR = 2.53 [95% CI: 1.96–3.26], p < 0.001). Interceptor® and Interceptor® G2 showed high blood-feeding inhibition against all strains. Conclusion Both free-flying laboratory bioassays (WHO Tunnel and I-ACT) consistently measured similarly, and both predicted the results of the experimental hut test. For bioefficacy monitoring and upstream product evaluation of ITNs in situ, the I-ACT may provide an alternative bioassay modality with improved statistical power. Interceptor G2® outperformed Interceptor ® against pyrethroid-resistant strains, demonstrating the usefulness of chlorfenapyr in mitigation of malaria. Graphical Abstract