Ornithonyssus bacoti, commonly known as the tropical rat mite, is a zoonotic ectoparasite that occasionally infests research rodent colonies. Most infestations have been attributed to wild rodents that harbor the mite and spread it to research animals, often during building construction or other activity that disrupts wild rodent populations. Although infestation may be clinically silent, severe outbreaks have been reported to cause pruritis, dermatitis, decreased reproductive performance, and anemia in rodents. In mid-2020, our institution experienced increased activity of wild mice, which were found to be infested with O. bacoti, diagnosed by microscopic exam and confirmed by fur swab PCR analysis. We elected to add O. bacoti to our quarterly health monitoring exhaust air dust (EAD) testing PCR panel, increase wild mouse control measures, and treat the environment with a sustained-release synthetic pyrethroid spray in an attempt to prevent colony animal infestation. Initial quarterly EAD health monitoring results in September of 2020 were negative for O. bacoti. However, in early 2021, multiple IVC racks tested positive for O. bacoti at quarterly testing. Treatment consisted of providing permethrin-soaked nesting material and surface spray treatment of the room and hallway with a sustained-release synthetic pyrethroid. Historically in the literature, O. bacoti outbreaks of research mice were not identified until mite burden was high enough to cause dermatitis on animal care workers. Due to modern molecular diagnostics and proactive PCR-based health monitoring surveillance, we were able to identify the outbreak earlier than would have otherwise been possible. To the best of our knowledge, this is the first report to successfully identify O. bacoti using environmental health monitoring PCR techniques. This outbreak demonstrates the importance of screening for O. bacoti in facilities with the potential for wild rodent infestation and highlights unique considerations when managing O. bacoti infestations. In addition, a novel permethrin-soaked enrichment item was developed for cage-level treatment.