Healthcare workers, including medical, dentistry and nursing students, have an increased risk of Mycobacterium tuberculosis infection. In this study the prevalence of tuberculin skin test (TST) positivity (defined as induration ≥ 5 mm) among healthcare students enrolled at the Sapienza University of Rome (Italy) in the years 2011-2012 was evaluated. Overall, 2,519 students with a mean age of 20.6 years were tested, of whom 62.4% were female, 0.4% were vaccinated with BCG and 4.6% were foreign-born. Eighty-two students (3.3%) were found to have a positive TST. Prevalence varied by age and geographical place of birth. A baseline evaluation of medical and other healthcare students' TST status at the beginning of their training is essential to better implement infection and control measures for tuberculosis infection.