In Egypt, buffaloes are considered as the second largest source of milk. Mastitis is a prime hindrance for milk production with severs economic losses, but scarce data are available about subclinical type in Egyptian buffaloes. The target of this study was to (a) Estimation of some biochemical alteration in mastitic whey milk and its role in milk quality and quantity. Mastitis caused an increase in total whey protein, immunoglobulin and albumin in cows' milk samples with sub clinical mastitis, while α–lactalbumin, and β-lactoglobulin showed significant decreased. There was a highly significant decrease in α -casein and қ casein in sub clinical mastitic samples in comparing with the normal milk. In addition, there were a significant decrease in β-casein and non-significant decrease in γ casein milk   protein fractions in sub clinical mastitic milk.  (b) investigate the major gram-negative bacteria linked with buffalo's mastitis, and (c) assess the antibiotic resistance pattern of the bacteria. To this end, 208 quarter milk samples were collected from 62 domesticated dairy buffaloes suffered from subclinical mastitis. Bacteriological assessment and biochemical methods, illustrated a total of 51 isolates (24.52%) represented different 10 Gram negative bacterial species. The most predominant ones were Klebsiella pneumoniae (25.49%), followed by Escherichia coli and Proteus mirabilis (13.73%) for each. The phenotypic antibiogram revealed that 50.9% of the isolated pathogens were resistant to three or more antibiotic groups. The most noticed resistance was versus ampicillin 43(84.31%) followed by both, tetracycline and trimethoprim–sulfamethoxazole 41(80.39%) for each one. Contrarily, the bacterial isolates were greatly sensitive to norfloxacin (68.6%), enrofloxacin (64.7%), then ciprofloxacin and amikacin (58.8%). On the other hand, the uniplex PCR, illustrated that 100% of the tested 13 Klebsiella pneumoniae strains carried bla ctx-m gene while 84.6% and 76.9% were positive to aacC and tet genes respectively., The implementation of high resistant associated Gram negative bacteria especially ESBL K. pneumoniae in subclinical mastitis considered a potential public health concern and requires good monitoring for achieving the efficient therapy.