Verticilactam and the new geometric isomers, verticilactams B and C, were produced by heterologous expression of the biosynthetic gene cluster for verticilactam using the Streptomyces avermitilis SUKA17 strain. Only verticilactam, a compound with a characteristic β-ketoamide unit within a 16-membered polyketide macrolactam conjugated with an octalin skeleton, had been previously reported having been isolated from Streptomyces spiroverticillatus JC-8444. In this report, minor verticilactam derivatives were isolated from the transformed strain, and their structures elucidated by spectral analysis. Verticilactam B was a geometric isomer at Δ17 and Δ19, and verticilactam C was the Δ19 and Δ21 isomer. In addition, the absolute configuration of verticilactam was confirmed by ECD analysis and NMR chemical shifts. The stereochemistry assignments of the hydroxy groups at C-10 and C-12 were supported by the domain organization of the polyketide synthase identified in the verticilactam gene cluster. Verticilactam showed moderate activity against the malaria parasite Plasmodium falciparum 3D7 strain with no significant cytotoxicity or antimicrobial effects.