Norway rats (Rattus norvegicus) carry several zoonotic pathogens and because rats and humans live in close proximity in urban environments, there exists potential for transmission. To identify zoonotic agents carried by rats in Baltimore, Maryland, USA, we live-trapped 201 rats during 2005–2006 and screened them for a panel of viruses, bacteria, and parasites. Antibodies against Seoul virus (57·7%), hepatitis E virus (HEV, 73·5%), Leptospira interrogans (65·3%), Bartonella elizabethae (34·1%), and Rickettsia typhi (7·0%) were detected in Norway rats. Endoparasites, including Calodium hepatica (87·9%) and Hymenolepis sp. (34·4%), and ectoparasites (13·9%, primarily Laelaps echidninus) also were present. The risk of human exposure to these pathogens is a significant public health concern. Because these pathogens cause non-specific and often self-limiting symptoms in humans, infection in human populations is probably underdiagnosed.