Susceptibility testing is one of the key tests in the therapeutic process of bacterial diseases, the accuracy in performance of the test and attention of physicians to the answer of this test can cause avoiding of bacterial resistance. In this study we evaluated the Accuracy of antibiotic susceptibility testing by disk diffusion method in Medical diagnostic Laboratories of Hamedan University of Medical Sciences in which the three Standards Bacteria; Escherichia coli, Staphylococcus aureus and pseudomonas aeroginosa were used. The disks of antibiotics from different companies in company with different disks from various laboratories for performance of susceptibility testing by disk diffusion agar method were prepared for Teaching Hospitals Laboratories of Hamadan. The accuracy of the laboratory work examined with a questionnaire with 39 questions. During this studying, 87.5% of laboratory staff didn’t have any attention to the effect of the antibiotics before use, 50% of laboratory personnel did not comply the medium standard diameter (4-5 mm), 62.5% of laboratories personnel did not fulfill the standard temperature incubator (35 ° C), 37.5% of staff not reported the genus of bacteria before performance of antibiogram test, 37.5% of laboratories personnel not prepared suspension in accordance with the 0.5 McFarland turbidity. In conclusion, our results show that to better carry out the testing, the laboratory personnel should be spend more time, also it is recommended that an incubator with 35 ° C for susceptibility testing should be prepared in the laboratory. Health center evaluate periodically the accuracy of antibiogram with preparation of bacteria and proper antibiotics in the laboratories.