Several bacteria exist in the air that pass through the ducts of air-conditioning systems. This study aimed to determine the effect of ultraviolet germicidal irradiation (UVGI) on six bacterial isolates (Serratia liquefaciens, Bacillus pumilus, Bacillus cereus, Staphylococcus lentus, Bacillus subtilis, and Oligella ureolytica) from the in-duct of an air-conditioning system. Bacterial species were exposed to UVGI with a wavelength of 264 nm for different durations. The exposed samples were then cultured and evaluated for growth. Growth was evaluated based on the colony-forming units, and the number of bacterial cells was estimated using spectrophotometry. Thereafter, the susceptibility value (Z value) to the UVGI was calculated for each bacterium. Each bacterium showed a different Z value under the same UVGI conditions, depending on the gram characteristics of the bacterium. The findings confirmed that UVGI is a potent technique for reducing the growth rate of multiple species of bacteria isolated from air-conditioning systems and this reduction depends on the structure of the bacteria (gram +ve and gram -ve) as well as exposure time. UV lamps can be placed in air-conditioning ducts to reduce or prevent the transmission of bacteria into the environment.