Epizootic shell disease (ESD) is an emerging form of shell disease of the American lobster (Homarus americanus) that has had detrimental effects on the fishery in southern New England. Three bacteria commonly isolated from lesions of wild lobsters with ESD—a novel Aquimarina sp. (A. ‘homaria’ I32.4), a novel Rhodobacteraceae species (‘Thalassobius’ sp. I31.1) and a Pseudoalteromonas sp. (Pseudoalteromonas ‘gracilis’ ISA7.3)—were applied directly to normal and abraded juvenile lobster carapaces, and then monitored for persistence over time and for the development of shell-disease lesions at 3 different temperatures (10°C, 15°C, and 20°C). Without abrasion of the carapace, no lesions developed in the exposures. After abrasion and exposure with a pure culture of A. ‘homaria’ I32.4, lesions developed at all 3 temperature and A. ‘homaria’ was detected in the lesions of all animals tested. Surprisingly, ‘Thalassobius’ sp. I31.1 also colonized these lesions. A coexposure with all 3 bacteria also demonstrated lesion development and the persistence of A. ‘homaria’ I32.4 and ‘Thalassobius’ sp. I31.1. The bacterium P. ‘gracilis’ ISA7.3 was not able to persist in any of the challenged lesions. Abraded areas of the cuticle with no bacteria added directly were also colonized by A. ‘homaria’ and ‘Thalassobius’ sp., and moderate lesions developed; however, the directly exposed lesions were significantly more severe (P < 0.05). The bacterium A. ‘homaria’, but not ‘Thalassobius’ sp., was detected in spontaneous lesions that developed independent of any abrasion and/or bacterial exposures. A novel bacterium, ‘Candidatus Kopriimonas aquarianus’ was also detected in spontaneous lesions. This study shows that 2 bacteria isolated from ESD lesions of wild lobsters are able to persist in and act together as important components of lesion development on abraded surfaces of American lobsters. This indicates that they are likely major contributors to lesion development in the ESD polymicrobial infection and may represent significant pathogens of the American lobster.