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Objective This study aimed at identifying demographic, socio-economic and tuberculosis (TB) exposure factors associated with non-compliance with the tuberculin skin test, the management and prevention of non-compliance to the test. It was carried out in the context of a survey of latent TB infection among undergraduate students taking healthcare courses in two universities in Salvador, Brazil, a city highly endemic for TB. Methods This is a cross-sectional study of 1164 volunteers carried out between October 2004 and June 2008. Bivariate analysis followed by logistic regression was used to measure the association between non-compliance and potential risk factors through non-biased estimates of the adjusted OR for confounding variables. A parallel evaluation of occupational risk perception and of knowledge of Biosafety measures was also conducted. Results The non-compliance rate was above 40% even among individuals potentially at higher risk of disease, which included those who had not been vaccinated (OR 3.33; 95% CI 1.50 to 7.93; p=0.0018), those reporting having had contact with TB patients among close relatives or household contacts (p=0.3673), or those whose tuberculin skin test status was shown within the survey to have recently converted (17.3% of those completing the study). In spite of the observed homogeneity in the degree of Biosafety knowledge, and the awareness campaigns developed within the study focussing on TB prevention, the analysis has shown that different groups have different behaviours in relation to the test. Family income was found to have opposite effects in groups studying different courses as well as attending public versus private universities. Conclusions Although the data presented may not be directly generalisable to other situations and cultural settings, this study highlights the need to evaluate factors associated with non-compliance with routine testing, as they may affect the efficacy of Biosafety programs., Article summary Article focus The reasons behind non-compliance with health monitoring are rarely investigated, even though high rates of non-compliance have been observed in several studies among groups ranging from the general population to students and healthcare professionals. Non-compliance with the tuberculin skin test (TST) may affect the efficacy of tuberculosis control programs. Key messages Having information on the targeted disease, as well as being at risk of this disease, was found to be insufficient to ensure compliance with routine testing. Non-compliance with the TST was associated with socio-economic status, gender and career choice, which suggests that cultural and psychological reasons for non-compliance are shared within such groups. Investigation of the reasons associated with non-compliance among different groups would be a first step to improve the efficacy of Biosafety programs. Strengths and limitations of this study This study was conducted only among healthcare students and within the context of a survey for latent TB infection. The risk factors found here to be associated with non-compliance may not be directly generalisable to other situations and cultural settings. This study is limited by the fact that all the information collected was self-reported, except for the frequency of non-compliance and the TST induration measurements. Unlike in other TST surveys, non-compliance due to logistics problems was addressed and minimised.