A total of 233 mastitic milk samples (151 cows and 82 ewes) and 90 mammary gland tissue samples (56 cows and 34 ewes) were collected from different Egyptian Governorates for P. multocida investigation as one of mastitis causing pathogens and its effect on the mammary gland tissues. The isolated P. multocidafrom clinical mastitic milk was slightly higher than that from subclinical form in both cows and ewes (15.3% versus 13.1% and 27.3% versus 26.7%, respectively) and it was isolated from the udder tissues of both cows and ewes with percentages of (17.85%) and (23.52%), respectively. Enzyme linked immunosorbent assay (ELISA) was used as confirmatory, rapid and reliable test for detection of P. multocida antibodies in the tested milk samples and they were detected in mastitic cow's and ewe's milk whey in (17.2%) and (34.2%), respectively. The antibiogram profile of P. multocida was studied for detection of its susceptibility to 15 different antibiotics to detect the drug of choice for its treatment. P. multocida isolated from cow's milk showed more resistance to various antibiotics than that isolated from ewe's milk. The DNA integrity of mammary gland tissue cells was detected using comet assay and the percentage of DNA damage was significantly elevated in case of P. multocidainfected mammary gland (P<0.05). In addition, the histopathological findings of P. multocida infected udder showed focal and/or diffuse chronic lymphocytic mastitis with an extensive degeneration and necrosis of the alveolar epithelium as well as interstitial tissue. Most of the mammary alveoli were filled with basophilic bacterial colonies with bipolar bodies positively stained by methylene blue and Giemsa stains. Cytological evaluation was conducted on all udder tissue samples and 16/18 positive cases (88.8%) were correlated with their histopathological examination. Histochemically, tissue sections from infected udder showed weak or no alkaline phosphatase activity and density of protein staining. It was concluded that P. multocida should be considered as an important sharing etiological agent of mastitis in both cows and ewes (especially in ewes) and associated with significant histopathological alterations in the glandular tissue structure. ELISA was considered as a quick and reliable technique for detection of P. multocida infection in the mammary gland especially in the un-vaccinated farms beside the traditional cultural method. The cytological interpretation was quiet helpful in rapid screening of the mammary gland affections.