Sierra Leonean Ebola survivors can have compromised health, with an increased mortality risk, so preventive health to minimize exposure to opportunistic and emerging pathogens is important for their clinical care and follow-up. The aim was to study the presence and temporal variation/distribution of opportunistic microsporidian species [Enterocytozoon bieneusi and Encephalitozoon spp. (E. intestinalis, E. hellem and E. cuniculi)] in chickens from different farms in Makeni city and its surroundings (Bombali District). Fresh faecal samples were collected from twenty chickens from Lion Poultry in April 2019, meanwhile twelve were collected in summer 2022 from this chicken farm and other private family farms across this district. Moreover, twenty-five water, twenty food, and three leaf samples that the chickens were using for eating/drinking were also collected in 2022. Faecal samples collected in 2019 were processed molecularly after extracting DNA using a Fast-Prep for Soil® kit, using a SYBR Green real-time PCR, meanwhile the most recent samples collected were tested only for Encephalitozoon species using a specific monoclonal antibody (Mab) of murine-origin IgG2a, patented as a diagnostic tool. Six faecal samples gave positive results microscopically (spores of Encephalitozoon spp. were observed in five samples and E. bieneusi in one). Five 2019 samples resulted positive by molecular methods: two E. intestinalis and E. hellem and one E. bieneusi; meanwhile, ten 2022 samples were positive for spores of the genus Encephalitozoon, specifically eight chickens and two water samples. None of the food samples were positive. To our knowledge, this is the first study reporting the presence of human-related microsporidia in chickens in Makeni city and its surroundings and is further confirmation of their presence in the water. The results also suggest a moderate circulation of Encephalitozoon spp. in chickens and farms, which could represent a risk for Sierra Leonean individuals living near these farms or a food risk.