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Background: Tuberculosis (TB) is an infectious disease caused by Mycobacterium species and its diagnosis, treatment, and management is vital. The existence of people with professional experience in this field is very important. Thus, the present study was conducted to recognize the quality of environmental microscopists working in Mycobacterium detecting laboratories in Isfahan Province, Iran, in 2015.Methods: This descriptive-analytical study was performed through a practical test on 27 individuals in 20 Environmental Laboratory Diagnosis of Pulmonary Tuberculosis in Isfahan Province. Five direct sputum smears with a related list were collected from each laboratory. The obtained data were analyzed.Findings: There was no significant relationship between quality of diagnosis and age, sex, employment status, exclusivity of the microscope, microscopic life, the microscopist’s activity in other sectors of the laboratory. However, university degree, work experience in the TB section, and the time that microscopists spend on each smear were effective on the quality of their diagnosis. Almost 75% of the participants scored high on the test.Conclusion: Since direct smear is still one of the most accessible and fastest methods of TB diagnosis, the diagnosis quality of these people should be evaluated at least annually by the Provincial TB Reference Laboratory.