Background: Healthcare-associated infections (HAIs) are a recognized public health problem worldwide. Point prevalence surveys (PPSs) can be used to measure the burden of all HAI types. Aim: To measure the prevalence of HAI and determine any changes in the epidemiology of HAI since the first Scottish national PPS. Methods: A national rolling PPS in National Health Service (NHS) acute, NHS non-acute, NHS paediatric and independent hospitals was carried out during September and October 2011 using the European Centre for Disease Prevention and Control protocol designed for the European PPS. The prevalence of HAI and distribution of HAI types were measured and the results compared with the first Scottish national HAI point prevalence survey of 2005/2006. Results: The prevalence of HAI was 4.9%, 2.5%, 6.1% and 1.2% in acute, non-acute, paediatric and independent hospitals respectively. The prevalence of HAI was significantly higher in acute hospitals compared with non-acute hospitals. There were no significant differences between the prevalence in the other hospital types. The prevalence of HAI in acute and non-acute hospitals was lower than the first survey by approximately one-third. The proportion of HAIs that were urinary tract infection, surgical site infection and bloodstream infection was higher and the proportion that were gastrointestinal including Clostridium difficile infection was lower in acute hospitals compared with the previous survey. Conclusions: The epidemiology of HAI has changed in Scotland since the first national survey in 2005/2006, thus infection prevention and control measures require to be refocused in this regard. The lower prevalence and changing epidemiology of HAI in acute and non-acute care suggest that there may be a temporal relationship with the implementation of the national programme of targeted HAI interventions in the intervening period.