A national point prevalence survey was undertaken over the period of one calendar year in Scotland from October 2005 to October 2006. The prevalence of healthcare-associated infection (HAI) was 9.5% in acute hospitals and 7.3% in non-acute hospitals. The highest prevalence of HAI in acute hospital inpatients was found in the following specialties: care of the elderly (11.9%), surgery (11.2%), medicine (9.6%) and orthopaedics (9.2%). The lowest prevalence was found in obstetrics (0.9%). The most common types of HAI in acute hospital inpatients were: urinary tract infections (17.9% of all HAI), surgical site infections (15.9%) and gastrointestinal infections (15.4%). In non-acute hospitals one in ten inpatients in two specialties (combined) - medicine (11.4%) and care of the elderly (7.8%) - was found to have HAI, and one in 20 inpatients in psychiatry (5.0%) had HAI. In non-acute hospital patients, urinary tract infections were frequent (28.1% of all HAI) and similarly skin and soft tissue infection (26.8% of all HAI). When combined, these two HAI types affected 4% of all the inpatients in non-acute hospitals. This is the first survey of its kind in Scotland and describes the burden of HAI at a national level.