The acquired and intrinsic resistance of bacteria to macrolide antibiotics limits the clinical application of these agents, and thus it is particularly important to discover novel macrolide antibiotics that can be administered to counteract the prevalence of bacterial resistance. In this study, we introduced some active 1,2,3-triazole side chains into the azithromycin at position 3-O, thereby obtaining a number of 3-O-substituted 15-membered azalides. Determination of the minimum inhibitory concentration (MIC) of these target compounds revealed that the compound 9g possessed the strongest antibacterial activity (MIC = 8-16 μg/mL) against drug-resistant strains and was generally 16- to 32-fold more active than the azithromycin (MIC ≥ 256 μg/mL). Combined analysis of the results of antibacterial activity together with theoretically calculated lipid/water partition coefficients (ClogP) indicated the importance of the chemical nature of the alkyl groups attached to the 1,2,3-triazole side chain in conferring promising antibacterial activity. The findings of molecular docking analyses indicated that compound 9g may bind to the A752 base of 23S rRNA in bacterial ribosome via hydrophobic or electrostatic interactions, resulting in the excellent antibacterial activity of this compound. Furthermore, the data of minimum bactericidal concentration revealed that compounds 9e, 9f, 9g and 9h are excellent bacteriostatic agents. In addition, the study of bactericidal kinetics confirmed that compound 9g is a time- and concentration-dependent agent.