Atrovimycin (1), a cyclodepsipeptide containing a unique vicinal-hydroxylated cinnamic acyl chain, was isolated and elucidated from Streptomyces atrovirensLQ13. The biosynthetic pathway of 1was achieved, revealing cytochrome P450 (Avm43) and epoxide hydrolase (Avm29) enzymes constructing the vicinal-dihydroxy substitution, as well as a tailoring P450 (Avm28) enzyme catalyzing β-hydroxylation of the l-Phe moiety. Atrovimycin shows in vitro antifungal activity and antitubercular activity against Mycobacterium tuberculosisH37Rv both in vitro(with MIC of 2.5 μg/mL) and in vivo.