Abstract Introduction The risk of tuberculosis (TB) in healthcare personnel (HCP) is related to its incidence in the general population. Healthcare students involved in clinical training could be exposed to occupational risks similar to those that HCP face. The prevalence of latent tuberculosis infection (LTBI) among undergraduate healthcare students with different working seniority in Italy was analysed. Methods A cross-sectional study under a screening programme for LTBI among undergraduate and postgraduate students attending Medical School at the Second University of Naples was conducted between January 2012 and December 2013 with clinical evaluations, tuberculin skin testing (TST) and, in positive TST students, Interferon-γ release assays (IGRA). Putative risk factors for LTBI were assessed by means of a standardised questionnaire. Results 3,374 students attending the Medical School of the Second University of Naples were submitted to a screening programme for TBC. 3,331 performed TST as a first-level test and 43 performed a Quantiferon test (QFT). 128 students were TST-positive and continued the diagnostic work with QFT, which was positive in 34 students. Of the 43 subjects who took the QFT as a first-level test only 1 was positive. In 35 students positive to the QFT test we formulated the diagnosis of LTBI by clinical and radiographic results. A correlation was found between age, non-Italian born persons, studying age, post-medical school status and LTBI. Conclusions The prevalence of LTBI among healthcare students in our study was very low. In countries with a low incidence of TB, the screening programmes of healthcare students can be useful for early identification and treatment of sporadic cases of LTBI.