The increasing use of illicit drugs imposes a public health challenge worldwide. People who inject drugs (PWID) are more susceptible to health complications due to immunosuppression associated with drug use and non-hygienic self-administration of substances, contaminants, and liquids. PWID are subjected to increased risk of acquiring and transmitting different pathogens (frequently functioning as sentinel cases for (re)emerging pathogens), including those transmitted by arthropods and vertebrate reservoirs in unhealthy environments. A clear association between injection drug use and HIV, HBV, and HCV infections has been described; however, other infectious viral and bacterial agents have been seldomly assessed. In this study, we investigated the seroprevalence of Bartonella spp., Coxiella burnetii, and Hantavirus among 300 randomly selected PWIDs from Rio de Janeiro, as part of a multi-city cross-sectional study carried out in the 1990s. Point seroprevalences and respective 95% CIs are as follows: 9.3% for C. burnetii (95% CI: 6.0%-13.0%), 1.0% for Bartonella spp. (95% CI: 0.0%-3.0%), and 4.0% for Hantavirus (95% CI: 2.0%-7.0%). In addition to the blood-borne pathogens, the results of this study increase our knowledge on other transmissible infectious agents in PWID. The high seroprevalence of C. burnetii and Hantavirus found among PWID is intriguing and suggests the need to carry out prospective studies, including molecular analyses, to confirm these findings and allow a better understanding of the putative relevance of these zoonotic infectious agents among PWID.