The anticipated worldwide surge in urban environments is generating ever-greater interest in the study of host–pathogen interactions in this specific type of habitat. We investigated the potential of city-inhabiting rodents to serve as the main Lyme borreliosis agents (Borrelia spp.) reservoir. We also tried to verify if anthropogenic disturbances changing the vertebrate species community composition may also alter the scheme of Borrelia spp. circulation. A total of 252 Apodemus mice (A. agrarius, A. flavicollis, A. sylvaticus) were captured in Warsaw (Poland), at sites classified into different zones of anthropogenic disturbance, ranging from suburban forests to municipal parks strictly in the city centre. Borrelia spp. infection, ascertained based on bacterium DNA presence in the rodents’ blood, was found only in A. agrarius and A. flavicollis (7.6 and 6%, respectively). Only one species from the Borrelia genus – the mammal-associated species B. afzelii – was found in the mice studied. We found no statistical evidence of a correlation between infection in Apodemus mice and the zone of anthropogenic disturbance where the mice were caught. Non-homogeneous concentrations of Borelia spp. infected specimens within the strict city centre area suggest a lack of contact between members of particular mice subpopulations, and their responsibility for relatively high, but local Borrelia spp. infection.