A One Health approach pilot study was carried out in selected villages within the Mnisi Traditional Authority’s area, Manyeleti Game Reserve, and Hans Hoheisen Wildlife Research Station in Mpumalanga Province, the Republic of South Africa from July to December 2018. The study’s main objectives were to identify positive and negative human-bat-environment interactions and microbiological screening of bats’ faecal samples for zoonotic fungi. Thirty-three purposively selected participants were asked to complete a structured questionnaire with multiple-choice and open-ended questions, and a total of 55 faecal samples were collected, 25 from identified bat roosting sites and 30 from captured bats. Ninety seven percent of respondents were aware of the presence or absence of bats in their immediate surroundings. However, the majority of them (87.9%) were uneasy about the presence of bats in their buildings, and nearly half (48.5%) were unsure whether bats play a positive or negative role in the environment. Some respondents (15.2%) stated that bats play beneficial roles in the environment, such as pollinating plants, spreading seeds of indigenous plants, catching harmful insects, and so on. More than half of the respondents (66.7%) stated that bats can be a nuisance; 18.2% of those polled reported contracting fungal diseases as a result of cleaning bat droppings without adequate protection. The analysis of faecal samples revealed that bats can harbour pathogenic fungi such as Aspergillus fumigatus, and A.flavus. We concluded that bats can harbour fungal pathogens that cause human diseases. Further research should be conducted to compile a complete list of fungi pathogens in bats in the study area.