BACKGROUND AND AIM: Physical and chemical cleaning to remove indoor microorganisms, which cause allergic reactions and respiratory diseases, is labor-intensive and time-consuming. An ultra-low volume (ULV) sprayer, a newly introduced device to inactivate pathogenic microorganisms, allows the disinfectant particles to reach hard-to-reach spaces indoors and is more cost-effective than the existing methods. However, few studies have been conducted to verify the efficiency of the ULV sprayer. We aimed to verify the disinfection efficiency of the ULV sprayer for inactivating total bacteria indoors, considering the factors affecting bacteria inactivation, and present the optimal ULV sprayer usage conditions to achieve the highest disinfection efficiency depending on room size. METHODS: Two-stage experiments were conducted. Total bacteria were grown in broth culture, seeded into biofilm templates, and placed in each location. VirkonTM Micro was dispersed into the rooms. Samples were collected pre-and post-spraying using Rodac plates to evaluate the disinfection efficacy. A response surface model was developed to optimize usage conditions for bacterial inactivation. RESULTS:The total bacteria removal efficiency was high (0.56 – 2.46 log10 reductions), including hard-to-reach spaces. The disinfectant concentration, spray amount, and room size had interactive effects on the total bacteria disinfection efficiency. The experimental data were fitted to a second-order polynomial model, with high coefficients of determination (R2) for all models (R2 0.82). The optimum conditions were a spray amount of 3.08 – 6.40 L in 160 m3, 3.78 – 7.22 L in 230 m3, and 5.68 – 8 L in 300 m3 when using dilution rates of 100 times. These conditions predicted a bacterial disinfection efficiency of 1.10 log10 reductions (92%) on all surfaces. CONCLUSIONS:Our results clearly indicate that the ULV sprayer effectively inactivates total bacteria indoors. The study results can be used as basic data for effective control of indoor surface microbes using ULV sprayer based on quantitative evidence. KEYWORDS: Built environment, Particle components, Microbes