Ornithobacterium rhinotracheale (ORT), is a bacterium that cause respiratory tract infection, has led to significant problems in the intensive poultry production. The current study aimed to isolate and identify ORT from broiler chickens, to detect antibacterial sensitivity and resistance of ORT isolates, and to test experimental infection of ORT in broiler chickens. One hundred eighty samples including tracheas, lungs and air sacs were subjected to isolation and phenotypic identification. Twelve suspected ORT isolates were used for molecular identification. Agar gel precipitation test was used to determine serotype of ORT isolates. Antibacterial sensitivity and resistance of ORT isolates were tested against 14 antibacterial drugs using standard disk diffusion technique. Pathogenicity of ORT was tested by experimental infection in broiler chickens. Results revealed that the incidence of ORT infection in broiler chickens in Assiut Governorate is 17.77% using isolation and phenotypic methods of identification, while it is 3.33% using molecular identification. Serological identification of ORT isolates indicated that all tested isolates, were belonged to serotype A. All ORT isolates were resistant to gentamycin, amoxycillin and cephradine with 100% incidence, where, 100% isolates were sensitive to colistin and doxycycline, 50% of isolates were sensitive to ampicillin and streptomycin, and 16.67% of isolates were sensitive to neomycin and trimethoprim. Living Newcastle attenuated vaccine, Lasota vaccine, exaggerates the clinical signs and lesions of ORT experimental infection.