The prevalence and antimicrobial susceptibilities of Staphylococci and Streptococci were assessed from subclinical mastitis cases. One hundred Coagulase-Negative Staphylococci (CNS) and 34 Streptococci were identified. The most frequently isolated species were Staphylococcus haemolyticus (27%) and Staphylococcus simulans (24%). Susceptible CNS species revealed the highest resistance to penicillin G (58%), ampicillin (48%), neomycin (20%), and oleandomycin (14%). CNS methicillin resistance rates within 82 isolates were 21.95% and 1.22% by disk diffusion and PCR methods, respectively. These results suggested the disk diffusion method was more prone to yield false positives. Partial sequencing of the 16S rRNA region from the mecA carrying isolate (S. haemolyticus) was homologous with S. haemolyticus sequences/accessions obtained from GenBank. However, the mecA gene sequence from this isolate was more closely allied with the S. aureus mecA gene of human origins. Identical sequence data was acquired from the National Center for Biotechnology Information (NCBI) database, suggesting horizontal gene transfer between the two species. CNS beta-lactamase activity within 81 isolates was 29.63%. The most frequently isolated Streptococcus species were S. uberis (52%) and S. agalactiae (15%). Oleandomycin was the least effective antimicrobial agent on these isolates with 59% susceptibility. Results indicated that CNS and Streptococci exhibited various antimicrobial resistance responses. Consequently, isolation and identification of udder pathogens in herds suffering from subclinical agents is essential to select the most effective antimicrobial agent. Moreover, multiple resistance features of methicillin resistant (MR) isolates should be considered during antimicrobial susceptibility tests.