During environmental monitoring of our institution's rodent watering systems, one vivarium was found to have high bacterial loads in the reverse-osmosis (RO) automatic water system. These findings prompted evaluation of the entire RO water production and distribution system. Investigation revealed insufficient rack and RO system sanitization, leading to heavy biofilm accumulation within the system. Approximately 2 wk after discovery in the water system, one of the bacterial organisms isolated in the water supply, Sphingomonas paucimobilis, was isolated from a peritoneal abscess of a severely immunodeficient B6. Cg-Slc11a1(r) Rag1(tm1Mom)/Cwi mouse housed in the same vivarium, suggesting that rodents drinking from this system were being exposed randomly to fragments of biofilm. Plans were developed to sanitize the entire system. Hypercholorination was used first, followed by treatment with a combination of peracetic acid and hydrogen peroxide. Between system sanitizations, a low-level chlorine infusion was added to the system as a biocide. Heterotrophic plate counts and bacterial isolation were performed on water samples obtained before and after sanitization procedures. We here discuss the process of identifying and correcting this important water-quality issue.