Background and aims: Acanthamoeba spp. and Balamuthia mandrillaris are free-living amoebas (FLA) that can severely affect individuals. FLA are naturally resistant to harsh conditions such as pH, temperature and chemicals. Although we have previously reported the circulation of Acanthamoeba spp. and B. mandrillaris in open-water systems from Leicester (UK), little is known about their presence in public drinking water supplies. Thus, the aim was to identify potential risks due to their presence in tap water. Methods: Twenty-five tap water samples were collected in duplicate in March 2022 from public restroom facilities, located in parks (14), shopping/leisure centres (7), bus (2) and train (1) stations and a cemetery (1). Taps were appropriately cleaned before and between collecting the water samples. Samples were centrifuged at 2500rpm for 30 minutes for spore recovery. Pellets were suspended in phosphate-buffered saline to extract DNA by disrupting the spores using the Fast DNA® Spin kit and purified. A triplex real-time TaqMan PCR assay was performed for the simultaneous detection of FLA species, using established methodologies. Results: All fifty samples tested for FLA were negative. The drinking water supplies in the taps of Leicester’s public restrooms are not sourced from storage tanks, which reduce the overall risk of the presence of FLA in tap water. However, limitations in the collection may have affected our results, as the incidence of Acanthamoeba keratitis in the UK is much higher than in the United States and Netherlands. This higher incidence has been linked to the use of water storage tanks, which would be in line with our preliminary results. Conclusions: Regular monitoring of tap water quality would be required at a wider scale in the city including households, public institutions and hospitals in order to rule out the risks for the presence of human-pathogenic FLA, particularly Acanthamoeba spp.