Antimicrobial resistance is a major threat for public health worldwide. Novel antimicrobial drugs are urgently needed to combat the growing prevalence of antimicrobial resistance. Nucleoside antibiotics represent a unique class of microbial natural products with distinctive structural features and diverse biological activities. We herein summarize recent findings on the biosynthesis of representative nucleoside antibiotics, and highlight recent advances in the discovery and rational generation of nucleoside antibiotics for the development of novel antimicrobial agents.