The purpose of this study was to estimate the prevalence of healthcare-associated infections (HAIs) in the region of Western Greece and its relationship with possible predisposing factors. Two 1-day prevalence studies were performed in all hospitals of the region. The average HAI prevalence was 2·9% (range 0–6·8%) in the hospitals and 0–22·7% between different medical wards. Overall, 90% of HAI patients had predisposing factors. The most frequently isolated microorganism was Escherichia coli (14·3%). The study revealed a relatively low overall point prevalence of HAI, but remarkable discrepancies between the hospitals and wards. This may be due to the presence of confounding medical conditions and/or underreporting of HAIs from certain hospital wards. Local point-prevalence surveys may increase the awareness of HAIs in hospital staff and contribute to the establishment of effective infection control.