Natural and agricultural areas are rapidly becoming urbanised, causing changes in habitat structure and diversity. Although the effect of urbanisation on the diversity of terrestrial plants and animals has been well studied, there is a significant gap in our understanding of how urbanisation impacts diversity in protists. Here, we measure the diversity of plasmodial slime moulds (a group of large, macroscopic protists also known as myxomycetes) in inner-city and semi-urban parks. We studied the impact of a range of environmental characteristics (pH, temperature, canopy cover, area of green space and substrate type) on species richness and composition of myxomycetes. We also examined the influence of different degrees of urban development surrounding these parks. Species composition was significantly different between substrate types but not between inner-city and semi-urban parks. Temperature was the only environmental characteristic that affected diversity, having a negative effect on myxomycete presence. Our findings suggest that myxomycete diversity in urban parks is driven by factors at the substrate level, and not by the park’s location within the city (inner city or semi-urban).