The prevalence of mastitis in breastfeeding Holstein Friesian cows is to be investigated. Culture-independent DNA-based techniques were used to analyze the isolated milk microbiota from Mastitic cows. Among the 133 cows evaluated, subclinical (SCM) and clinical mastitis (CM) afflicted roughly 13.5% (18/133) and 20.3% (27/133), respectively. The isolated microbiota was dominated by gram-positive bacteria like Escherichia coli and Staphylococcus aureus. A high throughput sequencing platform identified Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria as the most prevalent phyla of bacteria. The most often occurring genera were Bifidobacterium and Lactobacillus. In the same environment, Lactococcus, Acinetobacter, Staphylococcus aureus, Streptococcus agalactiae, and Coagulase-negative staphylococci-different bacterial species with higher potential roles in mastitis-were found. The scattering behavior of several samples in PCoA plots-beta diversity and Alpha diversity indices demonstrated the mastitis microbiota's considerable diversity. Season, lactation, and infection all affected the alpha diversity of the milk microbiota in Egyptian cows; four dominant phyla were found, and despite the presence of distinct bacterial communities in infected samples, mastitis did not significantly change alpha diversity. This study illuminates the prevalence of mastitis in Egyptian dairy cows, their microbiota, and risk factors for mastitis. The findings can reduce mastitis and improve dairy cow health and productivity.