The prevalence of latent tuberculosis (TB) infection (LTBI) and the incidence of active tuberculosis in healthcare workers (HCWs) in a Portuguese hospital were examined. This cross-sectional study comprises 4,735 hospital workers screened between May 2005 and September 2008. Tuberculin skin test (TST) and interferon-gamma release assay (IGRA) were used simultaneously in 1,219 HCWs (25.7%). Radiographs were taken in symptomatic HCWs or in test-positive HCWs. The tests were repeated annually or bi-annually depending on risk assessment. IGRA was positive in 32.6% and TST in 74.2% of the HCWs. Years spent in healthcare were a risk factor for a positive IGRA, but not for a positive TST. Repeated bacillus Calmette-Guérin vaccination increased the probability of TST+/IGRA- discordance (35.4% versus 54.4%, respectively). In those tested three times with TST during the study period (n = 59), the mean diameter of TST increased from 5 to 7 to 10 mm. Within 3 yrs, 31 HCWs were diagnosed with active TB (annual incidence rate 191 out of 100,000 people). In eight HCWs with active TB, TST and IGRA were performed at the time of diagnosis and each test was positive. TB burden in HCWs in Portugal is high. With IGRA, the number of radiographs needed to exclude active TB could have been reduced by about half without missing a case of active TB. Therefore IGRA should be introduced into TB screening programmes.