The aim of the current study was to establish the effects of preincubation application of low and high frequency ultrasound on egg weight loss, hatchability, supply organ weights, chick performance, and eggshell microbial activity in Japanese quail (Coturnix coturnix japonica). A total of 630 fresh eggs were randomly divided into 3 groups. Treatments were no ultrasound but eggs were sprayed with benzalkonium chloride solution (B), 35∈kHz ultrasound applied for 30 min (U35), and 130∈kHz ultrasound applied for 30 min (U130). At the beginning of the incubation, the eggs in the U130 treatment had lower coliform, Salmonella, and Staphylococcus counts than those in the B group. However, no significant differences were found in coliform, Salmonella, and Staphylococcus counts among treatments at d 14 of incubation. Among treatments, there were no significant differences in egg weight loss, hatchability, embryonic mortality, supply organ weights, spread of hatch, or relative growth.