Antimicrobials’ topical administration efficacy has not been assessed in dogs with upper respiratory tract disease. The aim was to compare the concentration of gentamicin in nasal lavage fluid (NALF) and in serum after three topical protocols. This was a prospective crossover study of ten healthy dogs. Gentamicin was nebulized for a duration of 1 week, twice a day, for 10 min in the first protocol (10-min protocol) and for 3 min in the second protocol (3-min protocol), while the third protocol consisted of the administration of 0.25 mL of gentamicin in each nostril (drop protocol). Median concentrations of gentamicin in NALF were 9.39 µg/mL (8.12–19.97 interquartile range), 4.96 µg/mL (4.60–6.43) and 137.00 µg/mL (110.5–162.00) in the 10-min protocol, 3-min protocol and drop protocol, respectively. The result for the drop protocol was significantly higher than those of both nebulization protocols in NALF (p = 0.039). In serum, the gentamicin concentration was 0.98 µg/mL (0.65–1.53) and 0.25 µg/mL (0.25–0.44) in the 10-min and 3-min protocols, respectively. Gentamicin was not detected in the serum of seven out of ten dogs in the drop protocol, and gentamicin was significantly higher in the 10-min protocol compared to the drop protocol (p = 0.001). This study found that the 10-min, 3-min and drop protocols achieved superior concentrations in NALF compared to the minimum inhibitory concentration for gentamicin-sensitive bacteria, while remaining below the toxic values in blood.