Black flies (Simulium spp.) are intermediate hosts and vectors of parasitic nematodes belonging to the genus Onchocerca (Filarioidea: Onchocercidae). Infection and subsequent transmission of infective third-stage larvae occur at the vertebrate host–skin interface. Experimental evidence presented here demonstrates that Onchocerca lienalis Stiles microfilariae orient to one or more components (microfilarial orientation factor[s]; MOF) in black fly saliva. MOFs may serve as a means for microfilariae to find and infect black flies during the act of blood-feeding. Directed movement through the host’s skin to the bite site is necessary because Onchocerca spp. microfilariae do not circulate in the blood. The substance directing microfilarial orientation appears to be a salivary protein, but it is not the Simulium vittatum Zetterstedt erythema protein (SVEP) described from New World Simulium spp. These results support earlier field observations that associated increased numbers of cutaneous microfilariae with black fly feeding and indicate that a fundamental molecular mechanism linked to vector saliva may be key for the maintenance of the life cycle of Onchocerca spp. Salivary molecules that induce orientation of microfilariae to the bite site are potential targets for use in transmission-blocking vaccines to uncouple this primary vector infection step.