1. Theoretical models suggest that increased vector species participation in pathogen transmission significantly increases the prevalence of vector and host infections. However, there has been a lack of empirical evidence to support this. 2. We linked transmission potential of multiple vectors species to observed patterns of enzootic pathogen transmission by conducting longitudinal field surveillance of West Nile virus (WNv) infections in Culex spp. mosquitoes and avian host communities in the southeastern U.S. We then used a temperature-dependent vectorial capacity model as a predictor of WNv infections in mosquitoes and birds using general linear mixed effects models. 3. Two WNv-competent Culex spp. mosquitoes were present in our study sites, Culex restuans Theobald during the spring and Culex quinquefasciatus Say during the summer. Empirical evidence of WNv transmission was limited exclusively to time periods when night time temperatures were suitable for accelerated within-vector viral replication, susceptible avian hosts (i.e. hatch year birds) were abundant, and Culex quinquefasciatus was the primary Culex spp. vector in the mosquito community. 4. Contrary to theoretical predictions, increased presence of competent vector species through time did not significantly increase the prevalence of infections in the WNv enzootic system. 5. Synthesis and applications. We extend a commonly used theoretical framework to quantify transmission potential of vector-borne diseases, the vectorial capacity equation, to account for species-specific variations in temperature-dependent development and host feeding preference. By quantifying key vectorial capacity parameters using field data from southeast United States we quantified the relative contribution of two Culex spp. species (Culex pipiens quinquefasciatus and Culex restuans) to the transmission of West Nile virus (WNv). Our findings suggest that to reduce the risk of human exposure to WNv in urban environments, vector control should focus on the primary WNv vector, the members of the Culex pipiens complex. Additionally, vector control may be more effective if it coincides with the onset of the avian breeding season, when most WNv amplification occurs. Moreover, our results highlight relevant knowledge gaps pertaining to WNv transmission by secondary mosquito species that coexist either in time or space with Culex pipiens complex mosquitoes. A better understanding of secondary WNv vector species is greatly needed in order to appropriately gauge their role in pathogen transmission dynamics.