A cross-sectional study was conducted on the performance of the tuberculin skin test (TST) and QuantiFERON^®-TB Gold In-Tube test (QFT-IT) for detecting latent tuberculosis infection among Thai healthcare workers (HCWs). Each HCW underwent both the TST and QFT-IT during the annual health screening. Among the 260 HCWs enrolled, the median age was 30 years (range 19–60 years), 92% were women, 64% were nurses and nurse assistants, 78% were Bacillus Calmette Guérin vaccinated, and 37% had previously taken the TST. Correlation between TST reaction size and the interferon-γ level was weak (r = 0.29; P < 0.001). Of the HCWs, 38% and 20% had a reactive TST and a positive QFT-IT, respectively. Using QFT-IT positivity as a standard for latent tuberculosis diagnosis, the cut-off for TST reactivity with the best performance was ≥13 mm with a sensitivity, specificity, false positivity, and false negativity of 71%, 70%, 30%, and 29%, respectively (area under the curve 0.73; P < 0.001). The independent factor associated with a false reactive TST was a previous TST (adjusted odds ratio 1.83; P = 0.04). Our findings suggest that the QFT-IT may be the preferred test among HCWs with previous TST. In settings where the QFT-IT is not available, appropriate cut-offs for TST reactivity should be evaluated for use among HCWs.