Microorganisms contamination in hatcheries has serious impacts on the quality and growth performance of ostrich. This study was conducted to examine the bacterial contamination of an ostrich hatchery environment and hatching eggs. Additionally, the inhibitory effects of some commercial disinfectants on microbial contamination on hatchery floor were also investigated. Our results indicated that, there were significant differences (P ˂ 0.05) among all microbial counts (Aerobic plate, Enteribactercae, Coliform) isolated from both walls and floors (eggs receiving room, setters, and Hatchers) and the floors of all sites were highly contaminated compared to the walls of all sites. Also, the floors and walls of Hatchers showed the highest microbial contamination, followed by the setters and lastly by the eggs receiving room. The overall prevalence of E. coli and Salmonella in all examine samples [ infertile, dead in shell eggs, hatching eggs, air, floor and walls swabs from the hatchery and chicks dropping (288) ] were 11.5 and 6.3% respectively. The most predominant serotypes of E. coli were O126:K71 (24.2%), O86:K61 (18.2%), and O128:K67 (15.2%), While, the most predominant serotypes of Salmonella were S. typhimurium (44.4%) and S. enteritides (33.3%). Our results indicated that the disinfectants had a significant effect (P ˂ 0.05) on microbial contamination and the best recommended concentration for effective control of microbial contaminants on Hatcher floor was 0.5% for Germicidan F1 and Viricidal Extra and 1.0% for Germicidan Iodes (for less than 2 hours). We results recommended that, the control programs should be maintained at hatcheries and breeder farms and should include routine microbiological monitoring and practical sanitation disinfectants to reduce the occurrence of such pathogens on hatching eggs and hatcheries.