Bovine mastitis, a growing issue in dairy farms, with Klebsiella species being associated with severe mastitis. This study was designed to evaluate the prevalence, biochemical changes, antibiogram susceptibility profile, and biofilm-producing capacity of Klebsiella pneumoniae isolated from Holstein cows with clinical mastitis at a private farm in Alexandria, Egypt. The overall isolation rate of Klebsiella species was 28%, which was further characterized by polymerase chain reaction (PCR) targeting the 16S-23S internal transcribed spacer gene as K. pneumoniae. Clinical mastitis significantly impacted milk composition and blood biochemistry, causing a decline in milk composition, reduction of antioxidant capacity, and increased enzymatic levels compared to normal controls. K. pneumoniae isolates showed 100% resistance to beta-lactam antibiotics, followed by resistance rates of 57, 43 and 43% to chloramphenicol, streptomycin, and sulfamethoxazole–trimethoprim, respectively, while ciprofloxacin and ceftriaxone showed complete susceptibility. The study found that all K. pneumoniae strains expressed the fimA, mrkA, and mrkD fimbrial genes, and the ecpA gene. The study highlights the high prevalence of multidrug-resistant and biofilm-producing K. pneumoniae strains in bovine clinical mastitis, emphasizing the need for improved antimicrobial usage and antibiofilm approaches to overcome the poor treatment response, as well as measuring antioxidant level and enzymatic activity in milk improved milk health screening.