Leptospirosis is a worldwide bacterial zoonosis which incidence is expected to increase in conjunction with global change. In urban ecosystems, synanthropic rats are the key source of Leptospira infection in humans and other animals. Risk assessment and prediction of human leptospirosis require investigations of the environment associated with the bacteria and infection patterns in the reservoir hosts. The objective of this study was to address the prevalence of mixed Leptospira infection in the lungs and kidneys of brown rats captured in three sites of the city centre of Vienna, Austria, between 2016 and 2018. A total of 96 brown rats were examined for the presence of Leptospira using PCR. Occurrence of mixed Leptospira infections was explored through next-generation sequencing (NGS). A logistic regression model was built to predict the individual infection status using morphological and land-use data. Overall, the prevalence of Leptospira interrogans in the kidney was 25% but varied among sites (0-36%). We did not evidence any pulmonary nor mixed infections. Host body mass and sex were strong predictors of Leptospira carriage in the sampled rats (relative variable importance (RVI) = 0.98 and 0.89, respectively) while the presence of water affected it moderately (RVI = 0.44). Our findings demonstrate that NGS is an unbiased approach to the direct characterisation of mixed leptospiral infections that could provide further insights into the ecology of Leptospira. Future surveillance programmes should consider the use of rats as sentinels for the early detection of emerging pathogenic Leptospira in urban ecosystems.