Human pathogenic free-living amoebae (FLA), specifically Acanthamoeba spp., Balamuthia mandrillaris and Naegleria fowleri, are rarely studied in animals’ gastrointestinal (GI) tract or their faeces as they do not have an obligate parasitic life cycle. However, FLA from different taxa have been recently recovered and identified in pigs’ GI tract and their faeces, suggesting a potential transmission source for pathogenic FLA and their associated intracellular bacteria. The main aim was to determine the presence of these three specific FLA species in faeces from cows and pigs monitored across Bombali and Tonkolili Districts, Sierra Leone (West Africa). Fresh faecal samples were aseptically collected, either from recent deposition or during defecation, from 12 pigs and 8 cows in Spring 2019. Samples were individually packed in plastic containers and immediately stored in a -80 °C freezer. Fourteen samples were collected from five locations across Makeni city (Bombali District): animal market (5 cows, 1 pig), general and pig slaughterhouses (5 pigs), Lorrey Park (2 cows) and Comforti (1 cow). Additionally, 6 pigs were monitored in Royanka, within the Tonkolili District. DNA was extracted from appropriately pre-concentrated faecal samples using FastDNA® Spin Kit. PCR inhibitors were removed using the QIAamp® micro DNA extraction kit. Extracts were screened for FLA using a triplex real-time TaqMan PCR assay which simultaneously identifies these three human pathogenic amoebae. Positive controls were used for each amoeba. All samples screened were negative. However, our results should be considered as inconclusive, owing to the limited number of animals and specific FLA species monitored. Moreover, we detected Acanthamoeba spp. in water reservoirs (wells and ponds) used for drinking by those animals from which samples were collected/screened. We also detected B. mandrillaris in the river in Royanka, which would confirm the presence of this emerging FLA in Tonkolili District, being the first time reported in the literature. In addition, the pre-concentration technique used might have not facilitated the detection of these FLA species, especially if they have a very low presence, as other recovery methods (e.g. filtration, sedimentation) have been described as more appropriate for recovering and culturing free-living protozoa from porcine faeces. Further monitoring studies, which also include non-pathogenic FLA taxa, would be required to understand the presence/circulation of these pathogenic/opportunistic species, particularly Acanthamoeba spp., in farm animals across these Sierra Leonean districts, to control the presence of foodborne pathogens.