OBJECTIVE To determine the number and proportion of healthcare worker (HCW) tuberculosis (TB) cases infected while working in healthcare institutions in the Netherlands and to learn from circumstances that led to these infections. DESIGN Cohort analysis. METHODS We included all HCW TB patients reported to the Netherlands TB Register from 2000 to 2015. Using data from this register, including DNA fingerprints of the bacteria profile and additional information from public health clinics, HCW TB cases were classified into 4 categories: (1) infected during work in the Netherlands, (2) infected in the community, (3) infected outside the Netherlands, or (4) outside these 3 categories. An in-depth analysis of category 1 cases was performed to identify factors contributing to patient-to-HCW transmission. RESULTS In total, 131 HCW TB cases were identified: 32 cases (24%) in category 1; 13 cases (10%) in category 2; 42 cases (32%) in category 3; and 44 cases (34%) in category 4. The annual number of HCW TB cases (P<.05), the proportion among reported cases (P<.01), and the number of category 1 HCW TB cases (P=.12) all declined over the study period. Delayed diagnosis in a TB patient was the predominant underlying factor of nosocomial transmission in 47% of category 1 HCW TB patients, most of whom were subsequently identified in a contact investigation. Performing high-risk procedures was the main contributing factor in the other 53% of cases. CONCLUSION In low-incidence countries, every HCW TB case should warrant timely and thorough investigation to help further define and fine-tune the HCW screening policy and to monitor its proper implementation. Infect Control Hosp Epidemiol 2017;38:976-982.