Background: The tuberculin test is widely used to identify tuberculosis infection. Some individuals infected with Mycobacterium tuberculosis may have an initial negative skin test reaction to tuberculin. The two step purified protein derivative skin test can decrease misinterpretation of tuberculin test .This study was aimed at comparing the two- step tuberculin skin test and booster phenomenon in health care workers and non hospital employees. Methods: One hundred and eighty five health service employees from Razi University Hospital and 181 non hospital employees were subjected to an initial tuberculin skin test. Those who were negative on the first test underwent a second one 2 weeks later. The reactions to the tests were measured 72 hours later. Tests with an induration of ≥ 10 mm was considered positive. Moreover, second tests with an induration of at least 6 mm increase relative to the relevant first tests were considered positive. Results: Compared to non hospital employees (n=79, 43.6%), a significantly higher number of health care workers (n=113, 61.1%) were positive on the first tuberculin test. 18.5 % of health care workers and 31.5% of non hospital employees demonstrated a boosted reaction after the second tuberculin test. The occurrence of boosted reaction was significantly associated with age both groups. There was no association between the presence of BCG scars and the occurrence of boosted reaction. Conclusion: the present study shows that the prevalence of TB was higher among health care worker than non hospital employees. Moreover, it demonstrated that non hospital employees had a higher age-associated booster reaction to the second tuberculin skin test than health care workers. Therefore, to avoid missing false negative cases, it would be necessary to do a second tuberculin test for subjects with a negative reaction to the initial test, especially in subjects older than 40 years.