To estimate the basic reproduction number (R(0)) of Borrelia lusitaniae and Borrelia afzelii, we formulated a mathematical model considering the interactions among the tick vector, vertebrate hosts, and pathogens in a 500-ha enclosed natural reserve on Le Cerbaie hills, Tuscany, central Italy. In the study area, Ixodes ricinus were abundant and were found infected by B. lusitaniae and B. afzelii. Lizards (Podarcis spp.) and mice (Apodemus spp.), respectively, are the reservoir hosts of these two Borrelia burgdorferi sensu lato (s.l.) genospecies and compete for immature ticks. B. lusitaniae R(0) estimation is in agreement with field observations, indicating the maintenance and diffusion of this genospecies in the study area, where lizards are abundant and highly infested by I. ricinus immature stages. In fact, B. lusitaniae shows a focal distribution in areas where the tick vector and the vertebrate reservoir coexist. Mouse population dynamics and their relatively low suitability as hosts for nymphs seem to determine, on the other hand, a less efficient transmission of B. afzelii, whose R(0) differs between scenarios in the study area. Considering host population dynamics, the proposed model suggests that, given a certain combination of the two host population sizes, both spirochete genospecies can coexist in our study area. Additional incompetent hosts for B. burgdorferi s.l. have a negative effect on B. afzelii maintenance, whose R(0) results > 1 only with high mouse population densities and/or low lizards abundance, but they do not seem to influence B. lusitaniae transmission cycle on Le Cerbaie. Secondly, our model confirms the importance of nymphs' infestation, of host population density and diversity, and spirochetes host association for the maintenance of the transmission cycle of B. burgdorferi s.l.