Background: Tuberculosis screening is part of the standard protocol for evaluating the risk of infection in healthcare workers. The aim of this study was to evaluate the prevalence of latent tuberculosis infection (LTBI) among students attending various healthcare profession degree courses and postgraduate medical courses at the School of Medicine of the University of Palermo, Italy, and assess the possible professional origin of infection. Methods: In total, 2946 students (2082 undergraduates and 864 postgraduates) took part in a screening program for LTBI between January 2014 to April 2019 using the tuberculin skin test (TST). Students with a positive TST result underwent a Quantiferon-TB test (QFT). Results: Among the 2082 undergraduates, 23 (1.1%) had a positive TST; the result was confirmed with QFT for 13 (0.62%) of them. Among the 864 postgraduate students, 24 (2.78%) had a positive TST and only 18 (2.08%) showed a positive QTF. Latent tuberculosis infections were significantly more frequent among postgraduates than undergraduates (2.08% > 0.62%, p < 0.0001). There was a higher number of subjects previously vaccinated for TB (18.87% > 0.24%, p < 0.0001), and of vaccinated subjects found positive for TST and QTF (66.67% > 7.69%, p = 0.001) in the postgraduate group. Conclusion: Latent TB is relatively low among medical school students in our geographic area. Nevertheless, this infectious disease must be regarded as a re-emerging biohazard for which preventive strategies are required to limit the risk of infection, especially among exposed workers.