Yersiniosis is a significant disease of economic importance in salmonids and non-salmonid fishes. However, few studies have investigated the pathogenesis, preventive, and treatment measures for this bacterial pathogen. Thus, the pathogenesis of Yersinia ruckeri in cultured Oreochromis niloticus in different localities was investigated throughout 2019. Besides, two experiments were conducted, the first feeding experiment based upon dietary supplementation by Ascorbic Acid and Difloxacine. In the second experiment, the supplemented fish groups were challenged by pathogenic Y. ruckeri strains, treated by Difloxacine, then the hematological, and immunological assays were analyzed. The supplemented fish groups by Ascorbic Acid and Difloxacine showed an increase in serum lysozyme, NO and SOD concentrations, phagocytic activity, and Interleukin IL-2 and IL-10 levels. The challenged fish by Y. ruckeri reported significant disturbances in transferases activities (ALT and AST), urea, total protein, and albumin concentrations. Moreover, RBC, Hb, PCV, nitroblue tetrazolium, lysozyme, NO and SOD concentrations, and Phagocytic activity were declined, with elevated interleukins level compared to the control. Thus, Ascorbic Acid supplementation and Difloxacine dosing could enhance the immune response, improve health status, and curtail the increased emergence of antibiotic-resistant bacteria.