Free-living amoebae (FLA), which are frequently found in the environment, include opportunistic pathogenic genera/species such as spp., , and . These pathogenic FLA are causative agents of amoebic encephalitis and keratitis in the case of genus and . In addition, amoebic infections are often related to contamination of domestic and recreational water sources. This study aimed to identify potentially pathogenic FLA in the hyperendemic area of human fascioliasis in a very-high-altitude area (3800-4100 m a.s.l.) of Bolivia and examine whether an association between both pathogens could be established from the environmental point of view. A total of 55 samples (28 soil and 27 water samples) were collected from various locations in the Northern Altiplano of Bolivia. Samples were processed by multiplex qPCR to detect the four pathogenic FLA genera/species. All samples were positive for the presence of , followed by spp. which was positive in 18 soil and 10 water samples. In contrast, was only detected in soil sources, whereas was not detected in any of the samples. The coexistence and diverse distribution of multiple FLA species in many locations at such a high altitude is worth mentioning and indicates a potential risk of coinfections. These findings suggest that FLA surveillance is a crucial factor to be considered when implementing preventive measures and improving public health in fascioliasis hyperendemic areas.