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This article describes laboratory research for the purpose of testing the efficacy of ozone treatment conditions to inhibit microbial growth in air-conditioning systems. Biofilm formation around condensation coils and drip pans takes place from collection of particulate organic matter combined with condensation of moisture in an air-conditioning unit. Ozone treatment was carried out periodically within an isolated section of air-handling ductwork encompassing the chilling coils and drip pan in a laboratory-scale air-conditioning system. After each treatment was completed, the spent ozone in the isolated section was exhausted to the outside atmosphere external to the controlled environment and/or could alternatively be scrubbed or neutralized prior to re-entry into the controlled environment. Efficacy was demonstrated at various combinations of ozone concentration, duration of exposure, and treatment frequency.