Staphylococcus aureus and Escherichia coli are main infection causing microbes. Both of the microorganisms also have great ability to develop resistant against antibiotics. Strains that have antibiotic resistance can cause infections in the form of epidemics. The objective of the present study was to identify and detect Escherichia coli and Staphylococcus aureus from samples of milk from mastitis positive buffaloes and cows and to assess the patterns of antimicrobial resistance profile. A total of 30 milk samples (buffalo: n= 15, Cows: n=15) were collected randomly from apparently healthy animals. Samples were collected from different dairy farms in the region of Rawalpindi. Samples were first checked for subclinical mastitis using surf field mastitis test (SFMT). Mastitis positive samples were analyzed in the laboratory by biochemical tests. Polymerase chain reaction (PCR) was used to for the molecular identification of Escherichia coli and Staphylococcus aureus utilizing the 16SrRNA gene. Antibiotic sensitivity test (AST) was performed to check antimicrobial resistance patterns of different antibiotics by using disk diffusion method. Out of 30 samples, 18 samples were found mastitis positive using SFMT (60%). For identification of Escherichia coli and Staphylococcus aureus API E20 and API Staph Kit was used respectively. Biochemical tests and PCR confirmed the presence of that out of 18 samples, 4 samples found for Staphylococcus. aureus and 6 samples were found positive for Escherichia coli. AST was performed to check antibiotic resistance patterns of isolated organisms and results have shown that both organisms were resistance against penicillin whereas Staphylococcus aureus was also found resistant against Oxytetracycline. Both organisms were also found highly sensitive to Ciprofloxacin and Erythromycin. The results of this study have shown that Escherichia coli and Staphylococcus aureus are present in selected dairy farms in Rawalpindi which can lead to their spread to other farms and cause a larger scale milk production loss.