In this paper, we develop a mathematical model that aims (1) to predict the distribution of negative ions generated by an air ionizer installed in a ventilation duct and (2) to predict the efficiency with which it inactivates bacteria. The transportation equation for the negative ions was resolved combined with the bulk air velocity and the electric field.The bacteria distribution was solved numerically by integrating the susceptibility constant, which was acquired from the experiments. Two types of bacteria (Serratia marcescens, Staphylococcus epidermidis) were aerosolized and released into a 9-m ventilation duct system. Inactivation efficiencies were calculated for inlet velocities from 2 to 6.5 m/s and for various ion intensities. The efficiencies for S. marcescens and S. epidermidis were 31.53% (SD, 11.4%) and 12.17% (SD, 0.43%), respectively, with susceptibility constants of 8.67 × 10 −11 Colony-Forming Units (CFU)/ions and 2.72 × 10 −11 CFU/ions, respectively. The modeling results matched those of the experiments well. The pressure penalty at the maximum velocity (6.5 m/s) was only 9 Pa. The results show that the use of negative ions has great potential to enhance indoor air quality by reducing airborne microorganisms in ventilation systems.