Facility-wide Corynebacterium bovis eradication was established using vaporized hydrogen peroxide (VHP) decontamination guided by C. bovis PCR surveillance. Prior attempts limited to culling PCR-positive mice and decontaminating affected rooms were ineffective in preventing recurrence. Because research aims often require trafficking to and use of procedural cores, a 12-mo facility-wide C. bovis PCR surveillance of 2064 specimens was performed and documented that, despite the presence of few clinically hyperkeratotic mice, 35% of the murine housing and use space was contaminated by C. bovis. The airways of IVC racks and air-handling units (AHU) provided a substantive niche for C. bovis survival, comparable to the primary enclosure, with 26% of murine and 22% of airway specimens PCR-positive for C. bovis. Equipment airway VHP sterilization in a 'flex room' required an 'active-closed' setting with the IVC rack connected to the AHU set to the VHP cycle, because 12% of specimens from 'static-open' VHP-exposed airways remained PCR-positive for C. bovis, whereas 0% of specimens from active-closed VHP exposures were positive. VHP decontamination of the 29,931-ft2 facility was completed in 2 mo. C. bovis PCR testing of IVC exhaust plenums for 200 d in previously C. bovis-affected rooms confirmed that none of the 259 specimens tested were PCR-positive for the organism. Monthly surveillance identified a single recurrence during June 2017 (month 9), ensuring rapid culling of C. bovis PCR-positive mice and acute VHP decontamination of equipment and rooms. Molecular persistence of C. bovis was resolved in procedural and personnel areas, and no murine or housing specimens tested C. bovis PCR-positive during study months 11 and 12. Furthermore, since the conclusion of the 12-mo study, none of the 452 additional murine, cell biologic, environmental, and monthly equipment surveillance specimens tested were C. bovis PCR-positive, documenting an 11-mo period of facility-wide C. bovis eradication to date. Study invalidation due to C. bovis can be avoided through PCR surveillance for the organism, immediate culling of PCR-positive mice, and acute VHP decontamination of affected areas.