Vicenistatin (<b>1</b>) is a potent polyketide antitumor antibiotic composed of a 20-membered macrolactam core appended to a unique aminosugar, vicenisamine. In this study, vicenistatin was isolated and its biosynthetic gene cluster identified from <i>Monodonata labio</i>-associated <i>Streptomyces parvus</i> SCSIO Mla-L010. A set of five genes, <i>vicC</i>, <i>vicD</i>, <i>vicE</i>, <i>vicF</i>, and <i>vicG</i>, was confirmed to be involved in the biosynthesis of the aminosugar by gene inactivations. VicG was characterized as an <i>N</i>-methyltransferase that catalyzes the methylation of the 4'-amino group in the last step of the aminosugar biosynthetic pathway; the <i>N</i>-demethyl intermediate 4'-<i>N</i>-demethylvicenistatin (<b>2</b>) was isolated from the Δ<i>vicG</i> mutant strain. In addition, <i>vicR1</i> was characterized as a positive pathway-specific regulatory gene. Notably, <i>N</i>-demethyl compound <b>2</b> was found to exert impressive antibacterial activities, with MIC values spanning 0.06-4 μg/mL, against a panel of Gram-positive bacteria including methicillin-resistant <i>Staphylococcus aureus</i>, Gram-negative <i>Helicobacter pylori</i>, and mycobacterium <i>Mycobacterium smegmatis</i> and the fungal pathogen <i>Candida albicans</i>. Compound <b>2</b> was also found to display reduced cytotoxicities relative to vicenistatin, especially against noncancerous human cell lines.