Abstract: Environmental issues are becoming the main cause of severe animal diseases, which greatly restricts the promotion of animal husbandry. Regular disinfection in animal farms is an important measure to prevent epidemic diseases. Due to the limitation of strong acidic electrolyzed water with a strong acidity (pH<2.7), corrosion of equipment, high cost and unsuitable application in practice, the spraying disinfection effects of neutral electrolyzed water (NEW, pH 6.5–8.5) for inactivating the bacteria in a layer breeding farms were investigated and compared with the commonly used chemical disinfectants (peroxyacetic acid and povidone-iodine). The effects of available chlorine concentration, treatment time and nozzle diameter on the bactericidal efficiency of NEW were also determined. The results showed that the bactericidal efficiency of NEW was higher than that of peroxyacetic acid and povidone-iodine under different treatment time. The bactericidal activity of NEW was increased with increasing the available chlorine concentration. NEW sprayed with a smaller nozzle diameter of 50 μm can improve the action efficiency of available chlorine and then increase the bactericidal effect for inactivation of bacteria in the air. Spraying NEW with available chlorine greater than 160 mg/L for maintaining 5 min (the fan turned off after spraying) is appropriate for disinfection in layer breeding houses. Therefore, NEW may be an alternative to chemical disinfectants used in spray disinfection in poultry farms, which will be play an important role in safety of animal production.