Recent research has found higher levels and longer total exposure of azithromycin, a macrolide antibiotic agent, in the interstitial fluid of the skin than in the plasma. This unique distribution is expected to contribute to its antimicrobial activity at the primary infection site. However, it remains unclear whether this characteristic distribution in the extracellular tissue space is common to macrolide antibiotics or if it is azithromycin-specific, with most macrolides largely localized intracellularly. In this study, we investigated pharmacokinetic characteristics of erythromycin and clarithromycin in the interstitial fluid of the skin of rats after intravenous drug administration, and compared the results with our previously reported results on azithromycin. Interstitial fluid samples were directly collected from a pore on the skin using a dissolving microneedle array. We found that the total macrolide concentrations in the interstitial fluid were significantly different among three macrolides. The rank order of the interstitial fluid-plasma concentration ratio was azithromycin (3.8 to 4.9) > clarithromycin (1.2 to 1.5) > erythromycin (0.27 to 0.39), and this ratio was stable after dosing, whereas higher drug levels in the skin tissue than in the plasma were observed for all three macrolides. Our results suggest that lower erythromycin concentrations in the interstitial fluid than in the plasma contributes to the emergence of bacterial resistance in the extracellular tissue space. Monitoring of total macrolide concentrations in interstitial fluid may provide valuable information regarding antimicrobial effects and the emergence of bacterial resistance for the development of an appropriate pharmacokinetics–pharmacodynamics-based dosing strategy.