Objectives: Thirty percent of the general population are Staphylococcus aureus nasal carriers. It has been shown that this increases with repeated contact with patients, but it is not known whether all categories of healthcare workers are at equal risk of carriage. We aimed to explore S. aureus nasal carriage among healthcare professionals. Methods: Prospective study conducted in two French university hospitals in 2014 and 2016. Volunteers were screened for S. aureus nasal carriage. Profession and hygiene habits were collected. Based on the results of this initial study, a second study focused on semi-skilled workers and biomedical equipment technicians (BETs) only; participants were given education on the basic rules of hygiene, then re-screened three months later. Results: In the initial study, 38.8% of the 436 participants were detected as nasal carriers. There was a significant difference in nasal carriage according to professional category (p < 0.0001); the lowest was found among administrative agents (17.3%), followed by healthcare providers (37.4%), laboratory technicians (37.6%). The greatest proportion was found among semi-skilled workers and BETs (52.9%). Spa-typing ruled out the hypothesis of a single clone dissemination among colleagues. After the three-month hygiene awareness campaign, all re-screened individuals remained positive, and with their respective initial strain. Conclusions: To the best of our knowledge we report here for the first time that semi-skilled workers and BETs are specifically more at risk of S. aureus nasal colonisation. This striking finding urges hospital hygiene departments to evaluate this specific professional category and implement strategies to improve hygiene awareness.