Globally, vessel strikes, entanglements, and ocean pollution negatively impact cetaceans by compromising skin integrity and health through direct injury or disease and by increasing susceptibility to chronic bacterial, viral, or fungal infections. Characterizing skin microbial assemblages in free-living cetaceans offers a means to evaluate and establish baseline trends in skin conditions. Currently, skin microbial data are collected only via biopsy sampling, which is a mildly invasive approach that represents only a small surface area of skin. In this study, we employed non-invasive environmental DNA (eDNA) flukeprint sampling of West Coast Transient killer whales in California and British Columbia to evaluate the method's effectiveness in detecting unique skin-associated bacteria. Using a paired approach, we found a greater number of bacterial exact sequence variants and bacterial genera unique to flukeprints compared with seawater controls. Functional pathways of unique flukeprint bacterial exact sequence variants were dominated by predicted functions likely related to DNA replication and repair, cell signaling, and protein folding. We compared bacterial results with visual skin condition assessments and other factors, finding that flukeprint bacteria are explained by a combination of geographic region, sampling month, whale age, whale sex, and the percentage of skin lesions visible in lateral photographs. This work demonstrates the applicability of eDNA flukeprint sampling for improving understanding of cetacean skin health, with implications for studying endangered or surface-elusive cetaceans that are not good candidates for biopsy sampling.