Corynebacterium bovis is a common pathogen in athymic nude mouse colonies. Control and eradication of the organism are challenging because depopulation and restricted colony access are often not options within vivaria. We evaluated potential sources and dissemination routes of C. bovis in an enzootically infected colony. Immunocompetent mice and personnel were evaluated for their potential to carry C. bovis, and husbandry and sanitation methods were evaluated for their efficacy in preventing cross-contamination. C. bovis was detected in furred immunocompetent mice previously exposed to infected athymic nude mice and in the nasopharynx of humans. Microisolation cages were not effective in maintaining athymic nude mice C. bovis-free when they were housed in a room known to contain immunodeficient mice with C. bovis infections. A tunnel washer that provided a ≥180 °F final rinse provided effective elimination of C. bovis from cage components. Passive and active air sampling techniques showed airborne dispersal of C. bovis despite the use of individually ventilated caging systems and stringent operational standards. Bacterial growth was not observed in settle plates placed inside autoclaved individually ventilated microisolation cages on various ventilated racks for 24-h periods. C. bovis aerosolization was shown to be a means of spread of the bacterium during cage-change procedures inside a class II type A2 biosafety cabinet. Our findings indicate that C. bovis can be a pervasive environmental contaminant in infected rodent holding rooms and successful eradication strategies must include environmental decontamination and attention to air quality.