The escalating threat of antimicrobial resistance demands innovative strategies to revitalize existing antibiotic classes. Here, we address the challenge of macrolide resistance by designing and synthesizing a series of novel azithromycin derivatives modified at the 4″-position with diverse aza-heterocycles (pyridine, quinoline, quinoxaline, pyrazine, indole) tethered through linkers of varying lengths. The resulting derivatives were evaluated against a panel of reference strains and clinically relevant bacterial isolates. Several compounds, particularly those bearing fused heterocyclic caboxamides attached with 3-aminopropylcarbamate linker to azithromycin (derivatives 5e-g), showed superior activity against Gram-positive strains, with 5f exhibiting an improved therapeutic index. Mechanistic investigations revealed that while derivatives 4f, 5a, and 5e-g displayed potent inhibition of bacterial translation, some of them also interfered with the normal function of bacterial DNA topoisomerases. Notably, these compounds retained activity against erm- and mef-mediated macrolide-resistant strains, demonstrating an ability to circumvent both ribosomal modification and efflux-driven resistance pathways. None of the conjugates induced the expression of the ermC reporter dependent on ribosome stalling at the ermCL regulatory region, an important therapeutic advantage underscoring the potential of azithromycin derivatives to overcome inducible mechanisms of resistance to macrolides. Taken together, these findings demonstrate that rational 4″-position heterocyclic modification of azithromycin can yield next-generation macrolides with enhanced antibacterial activity, dual mechanisms of action, and reduced susceptibility to prevailing resistance determinants.