The present study tested the biocidal effectiveness of the quaternary ammonium Hatching Egg Sanitizer Spray (HES Spray) in disinfecting broiler hatching eggs and in terms of its effects on eggshell permeability, water loss, and hatchability. The application of HES at a 1.5% or a 3.0% concentration resulted in significant reductions in the total aerobic counts on the egg surface of 98.1% and 99.9%, respectively, within 30 min of application. Molds and yeasts were significantly reduced by 3.0% HES at 14 days of incubation. Significant reductions in total aerobic counts on the egg surface due to 1.5% and 3.0% HES were also observed on eggs that were allowed to "sweat." The 3.0% HES concentration also reduced coliforms on egg surfaces. Hatchability of fertile eggs from a 32-wk-old flock was significantly increased, over 6.0%, by spraying 1.5% or 3.0% HES in comparison to controls that were not sprayed, with no significant difference in hatchability due to treatment observed in eggs from flocks that were 36, 42, 46, or 62 wk of age. This change in hatchability associated with spraying HES may be due to a change in eggshell permeability (respiration) caused by an interaction of HES with the eggshell cuticle.