The in vivo efficacy of rifampicin encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles was evaluated for the treatment of BALB/c mice experimentally infected with Brucella canis. The PLGA nanoparticles loaded with rifampicin (RNP) were prepared using the single emulsification-solvent evaporation technique, resulting in nanoparticles with a hydrodynamic diameter of 138 ± 6 nm. The zeta potential and polydispersity index values indicated that the system was relatively stable with a narrow size distribution. The release of rifampicin from the nanoparticles was studied in phosphate buffer at pH 7.4 and 37 °C. The release profile showed an initial burst phase, followed by a slower release stage attributed to nanoparticle degradation and relaxation, which continued for approximately 30 days until complete drug release. A combined model of rifampicin release, accounting for both the initial burst and the degradation-relaxation of the nanoparticles, effectively described the experimental data. The efficacy of RNP was studied in vivo; infected mice were treated with free rifampicin at concentrations of 2 mg per kilogram of mice per day (C1) and 4 mg per kilogram of mice per day (C2), as well as equivalent doses of RNP. Administration of four doses of the nanoparticles significantly reduced the B. canis load in the spleen of infected BALB/c mice. RNP demonstrated superior effectiveness compared to the free drug in the spleen, achieving reductions of 85.4 and 49.4%, respectively, when using C1 and 93.3 and 61.8%, respectively, when using C2. These results highlight the improved efficacy of the antibiotic when delivered through nanoparticles in experimentally infected mice. Therefore, the RNP holds promise as a potential alternative for the treatment of B. canis.