Acanthamoeba spp., an emerging opportunistic human parasite, has been previously detected in open-water systems across Leicester city and surrounding rural areas (Leicestershire, England), suggesting a potential risk for the population. 190 grass samples were collected over a year (June 2017 to August 2018) to study temporal variation, in and around Leicester city and adjacent rural areas, and to determine their circulation and risks. After appropriate pre-treatment to resuspend potential cysts/amoebas present in the grass avoiding potential soil contamination, DNA was extracted from each pre-concentrated sample using the Fast DNA® Spin kit. Acanthamoeba spp. was detected using a triplex real-time TaqMan PCR assay in 22 grass samples (11.57%). The seasonal study revealed the following prevalence percentages from Summer 2017 to Summer 2018: 15.7, 10.5, 13.1, 7.89 and 10.5%. To our knowledge, this is the first report reporting the presence and seasonal distribution of Acanthamoeba spp. in grass across Leicestershire. The moderate seasonal prevalence found would be logical owing to the high environmental resistance of their cysts. Moreover, although their interactions with wild animals is not well understood, a potential zoonotic reservoir might explain the moderate to high distribution found. Leicestershire’s citizens could be exposed when playing sports, or by contact of cornea or tissue wounds with the grass. Further genotyping studies are required for a better characterisation of the distribution and circulation of Acanthamoeba spp. in Leicestershire, and to identify appropriate public health interventions and decontamination techniques to minimise the risks identified, especially to protect individuals with their immune system compromised.