The risk of occupational tuberculosis (TB) infection and associated factors was estimated among all microbiology and pathology technicians and compared with a sample of nonclinical personnel in 17 Canadian acute care hospitals. Participants underwent tuberculin skin testing and completed questionnaires. Prior skin tests and vaccinations and all patients with TB hospitalized in the preceding 3 years were reviewed. Of the work areas where direction of air flow and air changes per hour were measured, only 51% were adequately ventilated. Among participating lab workers the average annual risk of tuberculin conversion was 1.0%. This was associated with lower hourly air exchange rates (16.7 versus 32.5 in workers with no conversion, p < 0.001) work in pathology (adjusted odds ratio [OR]: 5.4; [95% confidence interval: 1.3, 22], higher proportion of patients with missed diagnosis in the first 24 hours (per 20% increase-OR: 2.0; [1.3, 3.2], treatment delayed 1 week or more (per 20% increase-OR: 2.0; [3.2, 3.2]), and higher mortality (per 20% increase-OR: 2.5; [1.1, 5.6]). We conclude that laboratory workers, with no direct patient contact, have increased risk of tuberculin conversion in hospitals where a greater proportion of patients with TB die, or have delayed, or missed diagnosis, although this may be modified by workplace ventilation.