This research compared how bacterial-, plant-produced silver nanoparticles (Ag-NPs) and TH4 affected the eggshells microbial load and quail chicks' liver structure, embryonic mortality, and features related to hatchability. Ag-NPs were sensitized by bacterial and plant methods, and then identified by UV–visible spectroscopy, TEM, and FTIR spectroscopy. B-Ag-NPs were found in spherical shapes in size ranging from 7.09 to 18.1 nm versus multi-shape with size range of 25.0–78.1 nm for P-Ag-NPs. A total number of 624 eggs (in three equal groups) of Japanese quail flock were sprayed with TH4 as control, B-Ag-NPs and P-Ag-NPs. Thereafter, three eggs were sampled randomly from each group for determining important microbial groups. The remaining eggs were incubated according to the recommended incubation conditions. On the day of hatching, the percentages of hatchability and embryonic mortality were measured. Besides, five chicks from each treatment were slaughtered and the livers were utilized for ICP and histological tests. The effects of all three treatments on the microbial count in eggshells were comparable, according to the results. In addition, there was no negative effect on either hatchability percentage or embryonic mortality rate. The liver structure from both B-Ag-NPs and P-Ag-NPs treatments exhibited severe and moderate degeneration of hepatocytes, which may indicate possible hazardous effects of using nanoparticles. Using TH4 did not cause liver structure abnormality. In conclusion, using Ag-NPs for sanitizing hatching eggs effectively reduces the eggshell microbial count without affecting the hatchability percentage. Nevertheless, histological changes are appropriate to be considered as a safety parameter in Ag-NPs applications.