The behaviour of swimming pool users plays an important role in the prevention of chemical, microbial and physical risks associated with these recreational water environments. The aim of this study was to evaluate the hygiene-related behaviours and knowledge of users of indoor swimming pools in five Italian cities. A cross-sectional study was carried out using an anonymous self-administered questionnaire. The association between specific variables and patterns of behaviours and knowledge was assessed. A total of 4315 questionnaires were analysed. Of all respondents, 41.7% declared they had never read the swimming pool rules; 70.9% take a shower before entering the swimming pool; 13.5% said they urinated at least once in a swimming pool; 93.9% always wear proper footwear; 92.1% know what warts are and 69.3% know what mycosis is. A significant association was found between not reading the pool rules and unsafe behaviours, such as not showering (OR: 1.44, 95% CI 1.25–1.65), urinating in the pool (OR: 1.70, 95% CI 1.41–2.03) and not wearing any footwear (OR: 1.24, 95% CI 0.96–1.62). Male gender was significantly associated with urinating in the swimming pool (OR: 1.45, 95% CI 1.21–1.74) and not wearing footwear (OR: 1.71, 95% CI 1.32–2.23), but it showed to be protective against not showering (OR: 0.78, 95% CI 0.68–0.89). An age ≤ 18 years was a risk factor for not reading the pool rules (OR: 2.32, 95% CI 2.05–2.63), not wearing footwear (OR: 2.16, 95% CI 1.67–2.82) and not knowing what mycosis (OR: 7.04, 95% CI 5.34–9.27) and warts (OR: 7.94, 95% CI 6.86–9.20) are. Results reveal that incorrect behaviours are widespread among swimming pool users and that there is little awareness of the importance of reading pool rules for reducing health risks.