In wastewater systems in Kampala, Uganda, microbial contamination has increased over the past two decades. Those people who live or work along the Nakivubo channel and wetland and those who use the recreational areas along the shores of Lake Victoria are at an elevated risk of gastrointestinal infections. A quantitative microbial risk assessment (QMRA) was applied for five population groups, characterised by different levels of exposure to wastewater in the Nakivubo area, namely: (i) slum dwellers at risk of flooding; (ii) children living in these slum settlements; (iii) workers maintaining the drainage system or managing faecal sludge (sanitation workers); (iv) urban farmers; and (v) swimmers in Lake Victoria. The QMRA was based on measured concentrations of Escherichia coli, Salmonella spp. and Ascaris spp. eggs in wastewater samples. Published ratios between measured organism and pathogenic strains of norovirus, rotavirus, Campylobacter spp., pathogenic E. coli, pathogenic Salmonella spp., Cryptosporidium spp. and Ascaris lumbricoides were used to estimate annual incidence of gastrointestinal illness and the resulting disease burden. The QMRA estimated a total of 59,493 disease episodes per year across all 18,204 exposed people and an annual disease burden of 304.3 disability-adjusted life years (DALYs). Incidence estimates of gastrointestinal disease episodes per year were highest for urban farmers (10.9) and children living in slum communities (8.3), whilst other exposed groups showed lower incidence (<4.3). Disease burden per person per year was highest in urban farmers (0.073 DALYs) followed by sanitation workers (0.040 DALYs) and children in slum communities (0.017 DALYs). Our findings suggest that the exposure to wastewater is associated with public health problems, particularly children and adults living and working along the major wastewater and reuse system in Kampala. Our findings call for specific interventions to reduce the disease burden due to exposure to wastewater.