Acanthamoeba spp. are free-living amoebae that can cause life-threatening brain infections and can also infect the eyes and skin. Amoebas/cysts of this species have previously been isolated from the River Soar, which runs through the city of Leicester, England, and is frequently used by the public. It has also been found in grass samples collected from public parks and green spaces across the city. Acanthamoeba can host endosymbionts, which can include important human microbes, acting as Trojan horses. Although their zoonotic role is poorly understood, cysts of Acanthamoeba spp. and other free-living amoebae have been found in the faeces and/or the gastrointestinal tract of various animals, including domestic livestock (pigs, cows) and wildlife (squirrels, pigeons), suggesting a potential source of transmission. To investigate their zoonotic potential in pigeons, we analysed fresh droppings from 628 pigeons collected from various urban parks and green spaces in the city of Leicester and surrounding rural areas as follows: 136 samples in summer 2017, 140 in autumn, 124 in winter 2017/18, 135 in spring, and 94 in summer 2018. After appropriate pre-concentration, thin smears were stained with trichrome and examined by light microscopy. DNA was extracted using a Fast DNA® Spin Kit. PCR inhibitors were removed using the QIAamp® micro DNA extraction kit. The extracts were screened for Acanthamoeba spp. using a triplex real-time TaqMan PCR assay with an appropriate positive control. All samples tested microscopically and molecularly were negative. Although their zoonotic role is not well understood, a potential and unknown animal reservoir could explain the moderate to high distribution found in both the grass samples and the river, posing a potential risk to people involved in sports such as rowing, football, or rugby. Culture techniques should be used to better investigate the potential interaction of Acanthamoeba with urban pigeons.