Aims: This study was conducted to early detect the negative culture bacterial pathogens causing subclinical mastitis for the fast diagnosis of the disease and the reduction of some milk-transmitted pathogenic bacteria to human consumers. Methods and Results: A total of 171 positive California mastitis test (CMT) milk samples collected from asymptomatic dairy cows in Sharkia Governorate, Egypt were examined by conventional bacteriological methods. The obtained results revealed that Streptococcus species (77
2%), followed by Staphylococcus species (48
6%) and Escherichia coli (25
7%) were the most predominant bacterial pathogens isolated from positive culture milk samples, whereas Enterobacter and Pseudomonas species were the lowest ones (1
2%, for each). Herein, 13 (7.6%) negative culture milk samples were subjected to propidium monoazide (PMA) conventional PCR assay, followed by DNA sequencing of purified PCR amplicons. Sequence analysis identified seven different types of negative culture bacterial pathogens comprising as following; 4 Enterococcus hirae, 2 Bacillus cereus, 2 Staphylococcus aureus, 1 Bacillus mycoides, 1 Bacillus subtilis, 1 Enterococcus faecium and 1 Escherichia coli. Conclusions: All the detected negative culture bacterial pathogens by PMAPCR assay, followed by DNA sequencing were incriminated in causing subclinical mastitis disease and had serious implications on human public health through consumption of milk contaminated with those recovered bacterial pathogens. Significance and Impact of the Study: The used methods could be useful in the routine detection of negative culture bacterial pathogens present in milk and consequently, it will help in the rapid diagnosis of subclinical mastitis disease and the reduction of many milk-transmitted diseases to human.