Rat control programs in urban areas aim to reduce the risk of rat-associated zoonotic pathogens, with indiscriminate trapping and poisoning among the most commonly used rat control techniques. It has been shown, however, that anthropogenic disturbances to stable animal populations may have unpredictable effects on the ecology of zoonotic pathogens, and may paradoxically increase the risk of pathogen spillover. Urban Norway rats (Rattus norvegicus) are a reservoir for Bartonella spp. - a genus of zoonotic bacteria transmitted by hematophagous vectors, particularly fleas. Rats and fleas may be infected with more than one Bartonella species; however, mixed infections may be difficult to detect using culture and/or targeted molecular diagnostics. We developed a metagenomic approach to better characterize the Bartonella spp. present in rat blood and fleas collected in Vancouver’s Downtown Eastside neighbourhood. Results revealed that both B. tribocorum and B. vinsonii were circulating widely in the study population and that some fleas and rats were infected with both species. We then investigated the impacts of rat culling on Bartonella spp. carriage in the study area. We found that the odds of Bartonella spp. carriage increased in all city blocks except those in which culling occurred. This suggests that removing rats may prevent increases in Bartonella spp. prevalence, potentially lowering human health risks. Given that climate change is anticipated to increase the incidence and range of vector-borne zoonotic disease, it is vitally important that we determine the impact of rat control techniques on human health threats so that we can efficiently and effectively mitigate those threats in the future.