Subclinical mastitis (SCM) is an asymptomatic udder infection distributed worldwide that causes significant losses in the dairy industry. The study aims to detect the prevalence of this pathological condition and to identify the most prevalent related pathogens. A total of 440 quarter milk samples from 110 dairy cows were subjected to California mastitis test (CMT) and Modified Whiteside test (MWST) to quantify their efficacy in detecting subclinical mastitis in dairy cows. Quarter-wise prevalence of subclinical mastitis (SCM) was detected in 30.23% and 28.64% samples by CMT and MWST, respectively, while animal-wise prevalence of SCM was recorded in 60% and 55.45% by CMT and MWST, respectively. The left and right forequarter were most susceptible to SCM than other quarters. All positive samples by field tests were subjected to microbiological examinations. Staphylococcus aureus (S. aureus) (48.51%) which considered the primary pathogens among the bacterial isolates followed by Coagulase negative Staphylococci (40.09%), Escherichia coli (E.coli) (38.12%) and Streptococcus agalactiae ( S.agalactiae) (13.37%). The sensitivity and specificity of the CMT and MWST were 100%, respectively. The results revealed a strong association between these parameters and the diagnosis of subclinical mastitis in milk samples. In conclusion, the bacteria isolated from SCM play an important role on food poisoning especially S.aurus and E.coli.