Probiotics are routinely used in poultry husbandry due to health benefit on the host. The gut microbiota is now recognized to exert an important influence on the absorption and pharmacokinetics of many compounds. Therefore, this study was designed to evaluate the effect of candidate probiotics belonging to the species Lactobacillus brevis, L. plantarum and L. bulgaricus on pharmacokinetics of enrofloxacin in healthy chickens. The probiotic administration leads to higher degree of metabolism of enrofloxacin to ciprofloxacin in liver. The antibacterial drug was significantly faster absorbed (kab of 0.61 ± 0.54 h−1 and Tmax 7.81 ± 3.52 h) at lower concentrations (Cmax of 1.34 ± 0.18 μg·g−1) during the first 24 h of treatment in the probiotic's group. The values of kab, Tmax, and Cmax for the group, treated solely with enrofloxacin, were 0.10 ± 0.065 h−1, 15.42 ± 3.07 h, and 1.61 ± 0.24 μg·g−1, respectively. A significantly higher concentration of enrofloxacin and its metabolite ciprofloxacin in the liver was observed in the group with the probiotic treatment. Disposition of both drugs was not significantly changed in the duodenum and in the jejunum. The selected dose is appropriate for treatment of infections caused by pathogens with MIC < 0.06 μg·mL−1 irrespective of antibiotic administration alone or in combination with probiotics.