Spray-application of membrane-less acidic electrolyzed water (MLAEW) is a novel technique for disinfection in livestock houses. This study investigated the loss of free chlorine (FC, the major germicidal component in MLAEW) over distance during spray, as affected by air temperature and initial FC concentration. The antimicrobial effect of MLAEW on airborne bacteria from an aviary laying-hen house was examined. MLAEW was prepared with two FC concentrations (app. 15 and 60 mg L-1), and was sprayed at three air temperatures (18, 25, 32°C). The original MLAEW solution and MLAEW aerosols collected at 0, 25, and 50 cm from the spray nozzle were analyzed for FC concentrations. Bacteria were immersed into these MLAEW samples and numerated for viable count after 0.5-, 2-, and 5-min treatments. MLAEW aerosols collected at 0 cm lost 11.7 – 13.2% FC as compared to the original MLAEW solution. This initial loss was affected neither by the initial FC concentration (P = 0.13) nor by air temperature (P = 0.57). The rate of FC loss during travelling was 0.79 – 0.87 % per centimeter of aerosol travel distance (% cm-1) at 18°C, 1.08 – 1.15 % cm-1 at 25°C, and 1.35 – 1.49 % cm-1 at 32°C. This travelling loss was affected by air temperature (P = 0.02), but not by initial FC concentration (P = 0.38). Bacteria were completely inactivated in 0.5 min when treated with MLAEW samples with FC > 16.8 mg L-1, in 2 min when FC > 13.8 mg L-1, and in 5 min when FC > 7.2 mg L-1. Airborne bacteria from aviary hen house can be effectively inactivated by MLAEW with adequate FC concentration and contact time. During spray, antimicrobial efficacy of MLAEW aerosols decreased over distance due to FC loss which exacerbates at higher air temperature.