Mycobacterium bovis is responsible for bovine tuberculosis in both animals and humans. Despite being one of the most important global zoonotic disease, data related to the ecology and pathogenicity of bovine tuberculosis is scarce, especially in developing countries. In this report, we examined the dynamics of M. bovis transmission among dairy cattle in the Nile Delta of Egypt. Animals belonging to 27 herds from 7 governorates were tested by the Single Intradermal Comparative Skin Tuberculin (SICST), as a preliminary screen for the presence of bovine tuberculosis. Positive SICST reactors were identified in 3% of the animals spread among 40% of the examined herds. Post-mortem examination of slaughtered reactors confirmed the presence of both pulmonary and/or digestive forms of tuberculosis in > 50% of the examined animals. Targeted and whole-genome analysis of M. bovis isolates indicated the emergences of a predominant spoligotype (SB0268) between 2013–2015, suggesting a recent clonal spread of this isolate within the Nile Delta. Surprisingly, 2 isolates belonged to M. bovis BCG group, which are not allowed for animal vaccination in Egypt, while the rest of isolates belonged to the virulent M. bovis clonal complex European 2 present in Latin America and several European countries. Analysis of strain virulence in the murine model of tuberculosis indicated the emergence of a more virulent strain (MBE4) with a specific genotype. More analysis is needed to understand the molecular basis for successful spread of virulent isolates of bovine tuberculosis among animals and to establish genotype/phenotype association.