Cephalic recording cylinders enclosing craniotomy sites are used to facilitate long-term cerebral access in animals used in many disease models. These models can be hampered because infections caused by microbial pathogens can affect underlying central nervous system tissues. Typically, broad-spectrum antibiotic solutions are instilled into the cylinder to maintain asepsis within the chamber. Despite prophylaxis against antimicrobial pathogens, bacterial and mycotic organisms have been cultured from chambers of recording cylinders used in a laboratory colony of rhesus macaques (Macaca mulatta). In an attempt to develop a more effective maintenance protocol, preparations of chlorine dioxide were evaluated for their ability to control microbial contamination. Chlorine dioxide test solution was infused into the chambers, and samples for microbial culture were collected throughout several months to evaluate microbial efficacy. Although effective against bacterial isolates in vitro, chlorine dioxide solution failed to adequately control these organisms in vivo. However, 1 or 2 infusions of the solution did eliminate mycotic organisms in most macaques. In contrast, antibiotics inhibited bacterial growth, but allowed fungal proliferation. A treatment schedule that alternated the use of antibiotic solutions and chlorine dioxide solution proved to be an effective means of controlling microbial contamination of recording cylinder chambers in this rhesus macaque colony.