Hatcheries are hubs for incoming eggs and progeny flock output, making them a crucial component of the poultry production chain. This study involved performing quantitative microbiological tests in a commercial hatchery with numerous compartments, including an egg handling room, cold storeroom, setter room, hatcher room, and chick production hall. There were 150 air samples and 180 surface swabs collected in the incubator before and after disinfection over ten visits, in addition to 250 yolk sac and organ samples taken from late-dead embryos. As a result, surface swabbing could detect higher contamination levels than open-plate methods in all hatchery visits, mainly in handling eggs, cold storage, and hatcher halls. This study examines the presence of Pseudomonas aeruginosa strains in hatchery environments and dead embryos. Biochemical and PCR testing were used to identify P. aeruginosa using 16SrDNA primers at 956 bp and the toxA gene at 396 bp. In hatchery environmental samples, the incidence rate was 10.7%, and in dead embryos, it was 10%; therefore, maintaining good hygiene, especially in hatcheries, is essential for Pseudomonas species control. Subsequently, in this study, a virulent strain of P. aeruginosa was subjected to an in vitro test with 10 disinfectants from six chemical groups. Iodine compounds with phosphoric acids, per-acetic acid, sodium di-chloroisocyanurate, and quaternary ammonium compounds (QACs) with glutaraldehyde compounds showed 100% microbial reduction even in the presence of organic matter with exposure times of 30 min. It was concluded that the most effective and cost-effective way to prevent and control infections is to use a disinfectant with sufficient concentration and exposure time in hatcheries.