The development of interventions that reduce Lyme disease incidence remains a challenge. Reservoir-targeted approaches aiming to reduce tick densities or tick infection prevalence with Borrelia burgdorferi have emerged as promising ways to reduce the density of infected ticks. Acaricides of the isoxazoline family offer high potential for reducing infestation of ticks on small mammals as they have high efficacy at killing feeding ticks for a long period. Fluralaner baits were recently demonstrated as effective, in the laboratory, at killing Ixodes scapularis larvae infesting Peromyscus mice, the main reservoir for B. burgdorferi in northeastern North America. Here, effectiveness of this approach for reducing the infestation of small mammals by immature stages of I. scapularis was tested in a natural environment. Two densities of fluralaner baits (2.1 baits/1,000 m² and 4.4 baits/1,000 m²) were used during three years in forest plots. The number of I. scapularis larvae and nymphs per mouse from treated and control plots were compared. Fluralaner baiting reduced the number of larvae per mouse by 68% (CI95: 51–79%) at 2.1 baits/1,000 m² and by 86% (CI95: 77–92%) at 4.4 baits/1,000 m². The number of nymphs per mouse was reduced by 72% (CI95: 22–90%) at 4.4 baits/1,000 m² but was not significantly reduced at 2.1 baits/1,000 m². Reduction of Peromyscus mouse infestation by immature stages of I. scapularis supports the hypothesis that an approach targeting reservoirs of B. burgdorferi with isoxazolines has the potential to reduce tick-borne disease risk by decreasing the density of infected ticks in the environment.