Armeniaspirols (1-3) are potent antibiotics against Gram-positive pathogens. Through a biosynthetic investigation, we identified four enzymes involved in the structural modification of 1-3. Manipulation of their activity led to the generation of 4-6 and nine novel analogues, 7-15. Bioactivity assessments revealed that the pyrrole chloro group and the methyl group are important for the antimicrobial activities of armeniaspirols, which lays the foundation for future structure optimization and mechanism of action studies of armeniaspirols.