Free-living amoebae are ubiquitous protozoa, frequently found in the aquatic environment. Acanthamoeba spp., in some conditions, causes amoebic keratitis. Our research project aimed at studying in vivoAcanthamoeba spp. that are possibly present in water destined for human consumption. Thus, we can evaluate the rate of water contamination by determining the critical areas of its presence. In total, 150 water samples were analysed from Rabat. All the samples were collected from five different sources: river, fountain water, seawater, public bath water and tap water. The samples were distributed over three seasons: spring, summer and autumn. The positive samples by culture method were confirmed by polymerase chain reaction (PCR) method. The obtained results by microscopic identification and PCR method showed a high percentage of the presence of Acanthamoeba spp. in water in Rabat. However, during the sampling period, we noticed a non-uniform division of the positive samples with a remarkably high rate during summer. Our study showed that water contamination by Acanthamoeba spp. in Rabat, Morocco is at high risk of having a negative impact on public health. It is necessary to do a follow-up and study the health impacts to better evaluate the risk associated with this contamination by Acanthamoeba spp.