Staphylococcus aureus (S. aureus), the bacteria most frequently associated with mastitis in cattle and buffalo, has a large number of genes connected to antibiotic resistance. The objective of the current research was to determine the erythromycin and macrolide resistance of S. aureus isolated from mastitic milk of cattle and buffalo, particularly resistance-related genes (ermA, ermB, ermC, ermT, and msrA). Therefore, 150 dairy cattle and 135 dairy buffalo bred by small farmers in various governorates of Egypt (Cairo, Giza, Kalyobia, Fayoum, and Kafr El-Sheikh) provided a total of 285 milk samples. Inspection revealed that a total of 34 (22.7%) and 36 (26.7%) milk samples from cattle and buffalo, respectively, had clinical mastitis. With a total recovery of 31 (44.3%) S. aureus isolates. Bacterial isolation and identification of S. aureus verified the isolation and identification of 14 (41.2%) and 17 (47.2%) S. aureus isolates from cattle and buffalo, respectively. Utilizing a TaqMan probe-based real-time PCR method that targets the nuc gene, all S. aureus isolates were verified. In instances of bovine mastitis in India and Kenya, conventional PCR targeting the nuc gene, followed by DNA sequencing and phylogenetic analysis, revealed a high homology (100%) with that of S. aureus strains isolated from milk. For the tested genes, the prevalence of resistant strains was 9.6% (ermA), 64.5% (ermB), 70.9% (ermC), 19.3% (ermT), and 9.6%. (msrA). Therefore, effective control measures should be adopted to stop the spread of drug-resistant S. aureus to humans.