Tiamulin is an antibiotic approved exclusively in veterinary medicine, active against G-positive bacteria as well as Mycoplasma spp. and Leptospirae spp. The study was aimed to establish its pharmacokinetics and to evaluate drug effects on resistance in cloacal flora in vivo in geese. Eight healthy geese underwent to a two-phase longitudinal study (60 mg/kg single oral administration vs 60 mg/kg/day for 4 days) with a two-week wash-out period. Blood samples and cloacal swabs were collected at pre-assigned times. Minimal inhibitory concentration (MIC) has been evaluated for each isolated bacterial species. The pharmacokinetic parameters that significantly differed between the groups were Cmax (p = 0.024), AUC0-t (p = 0.031), AUC0-inf (p = 0.038), t1/2kel (p = 0.021), Cl/F (p = 0.036), and Vd/F (p = 0.012). Tiamulin exhibited a slow to moderate terminal half-life (3.13 h single; 2.62 h multiple) and a rapid absorption (1 h single; 0.5 h multiple) in geese, with an accumulation ratio of 1.8 after multiple doses. An in-silico simulation of multiple dosing did not reflect the results of the in vivo multiple dosage study. In both treatments, the MIC values were very high demonstrating a resistance (> 64 μg/ml) against tiamulin that can be present prior the drug administration for some strains, or emerge shortly after the commencing of treatment for some others.