Canustatins A and B, two structurally novel polyketide metabolites, were isolated from the actinobacterium Streptomyces canus NA07887. Their structures were determined through comprehensive spectroscopic analysis, including NMR and HRESIMS, combined with the comparison of experimental and theoretical electronic circular dichroism (ECD) spectra. Canustatin A features an unprecedented 3,6-dioxabicyclo[3.1.0]hexane moiety, a structural motif rarely encountered in type I polyketides. Stable isotope labeling studies facilitated the proposal of a plausible biosynthetic pathway for these compounds. In addition, canustatin A exhibited moderate antibacterial activity against multidrug-resistant Acinetobacter baumannii ATCC AYE.