Bovine mastitis, an inflammation in dairy cattle, causes significant economic losses, often triggered by bacterial pathogens like Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). The overuse of antibiotics has led to resistance through β-lactamase enzymes, including extended-spectrum beta-lactamases (ESBLs). This study focuses on isolating and characterizing ESBL-producing E. coli and K. pneumoniae from Egyptian dairy farms using both culture and molecular methods. Among 259 subclinical and 127 clinical mastitis milk samples, Enterobacteriaceae was found in 79.15% and 79.53% of cases, respectively. E. coli was the predominant pathogen, with 141 subclinical and 77 clinical cases, while K. pneumoniae was less common. The study identified ESBL-producing strains in 71 E. coli and 25 K. pneumoniae from subclinical mastitis, and 36 E. coli and 15 K. pneumoniae from clinical mastitis. The blaTEM gene was found in 85.04% of E. coli and 100% of K. pneumoniae isolates, indicating its dominant role in resistance. Antibiotic sensitivity analysis revealed significant resistance, with E. coli showing 100% resistance to tetracycline, 77.6% to ciprofloxacin, and 46.7% to gentamycin, while K. pneumoniae exhibited 100% resistance to tetracycline, 70% to ciprofloxacin, and 62.5% to cefpodoxime. Both pathogens had lower resistance to amoxicillin-clavulanic acid. These results underscore the need for improved control measures to combat antibiotic resistance and highlight the importance of alternative therapeutic strategies in treating mastitis.