Abstract Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer progression. Evidence suggests colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we show that colibactin-derived ICLs are unstable toward depurination and elimination of the 3′ phosphate. This pathway leads progressively to the formation of nicks SSBs and cleavage DSBs and is consistent with the earlier determination that non-homologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein further our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.