There is an increasing need to evaluate the impact of chemotherapeutic and vector-based interventions as onchocerciasis affected countries work towards eliminating the disease. The Esperanza Window Trap (EWT) provides a possible alternative to human landing collections (HLCs) for the collection of anthropophilic blackflies, yet it is not known whether current designs will prove effective for onchocerciasis vectors throughout sub-Saharan Africa. EWTs were deployed for 41 days in northern Uganda and south eastern Tanzania where different Simulium damnosum sibling species are responsible for disease transmission. The relative efficacy of EWTs and HLCs was compared, and responses of host-seeking blackflies to odour baits, colours, and yeast-produced CO2 were investigated. Blue EWTs baited with CO2 and worn socks collected 42.3% (2,393) of the total S. damnosum s.l. catch in northern Uganda. Numbers were comparable with those collected by HLCs (32.1%, 1,817), and higher than those collected on traps baited with CO2 and BG-Lure (25.6%, 1,446), a synthetic human attractant. Traps performed less well for the collection of S. damnosum s.l. in Tanzania where HLCs (72.5%, 2,432) consistently outperformed both blue (16.8%, 563) and black (10.7%, 360) traps baited with CO2 and worn socks. HLCs (72.3%, 361) also outperformed sock-baited (6.4%, 32) and BG-Lure-baited (21.2%, 106) traps for the collection of anthropophilic Simulium bovis in northern Uganda. Contrasting blackfly distributions were observed on traps in Uganda and Tanzania, indicating differences in behaviour in each area. The success of EWT collections of S. damnosum s.l. in northern Uganda was not replicated in Tanzania, or for the collection of anthropophilic S. bovis. Further research to improve the understanding of behavioural responses of vector sibling species to traps and their attractants should be encouraged.