In Egypt, the common tick species on cattle and equines are Rhipicephalus annulatus and Hyalomma excavatum, both vectors of tick-borne diseases (TBD). This study aimed to characterize of R. annulatus and H. excavatum using morphological, molecular, and immunological tools, and to investigate the tick-borne pathogens (TBP) associated with these species. A total of 186 adult ticks were collected from Giza, Sohag, New Valley, and Faiyum, including 28 ticks from 2 horses and 158 ticks from 104 cattle. The ticks were characterized through light microscopy (LM), scanning electron microscopy (SEM), and polymerase chain reaction (PCR) coupled with sequencing, along with molecular screening of associated pathogens. Blood samples were also collected from cattle and three calves as a negative control for immunological characterization. The integrated analyses confirmed R. annulatus as the dominant tick on cattle in all regions and on horses in Giza, while the tick H. excavatum was dominant on cattle in New Valley. Infection rates for anaplasmosis and borreliosis were 42.5% and 11.8%, respectively. Novel potential Ehrlichia sp. and Borrelia theileri genotypes were detected in both tick species. Western blotting analysis supported the development of a universal anti-tick vaccine, with detected bands reacting in the same molecular weight range (99–100.4 kDa) in both species. Combining different characterization methods shows promise for accurately diagnosing and controlling ticks and TBD and updating the understanding of native or exotic threats.