This study presents the first molecular detection and characterization of protozoa associated with livestock abortion, including Toxoplasma gondii, Neospora caninum, Sarcocystis spp., and Microsporidia spp., in the South Sinai Governorate, Egypt. A total of 226 blood samples were collected from cattle, goats, and sheep with a history of abortion. Molecular analysis was conducted using PCR and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. The results revealed the prevalence of T. gondii in cattle at 12.33% (9/73), while goats and sheep tested negative. In contrast, 14.92% (10/67) of goat samples tested positive for Microsporidia spp., with no cases detected in cattle or sheep. No infections with Sarcocystis spp. or N. caninum were identified in any tested animals. Across all examined livestock, the overall prevalence of T. gondii was 3.98% (9/226), while Microsporidium spp. exhibited a slightly higher prevalence at 4.42% (10/226). Molecular sequencing identified three novel T. gondii strains in cattle recorded in the GenBank database under accession numbers MZ197902, MZ197903, and MZ197904, showing 99.9% genetic similarity to reference strains. Additionally, three novel strains of Enterocytozoon bieneusi (MZ197781, MZ197782, and MZ197783) were detected in goats. Phylogenetic analysis indicated a close evolutionary relationship between these newly identified E. bieneusi strains and known genotypes, suggesting a high degree of genetic conservation. These findings underscore the ongoing risks of T. gondii and E. bieneusi to livestock and public health. Continued surveillance is necessary to assess the zoonotic potential of these novel strains to aid in effective management strategies.