Groundwater is consumed by over 2 billion people globally, though it can be impacted by microbial and chemical contamination in both rural and (peri-)urban areas. This issue is particularly pertinent in regions like East Africa, where rapid urbanisation has strained local infrastructure, including water and sanitation systems. We use selected tracers of human and animal waste to assess the quality of community drinking sources with regards to surface-derived groundwater inputs and to compare urban versus rural water quality, under the rapidly developing urban area of Gulu, Northern Uganda. Specifically, we examine bulk and fluorescent dissolved organic matter (DOM), microorganisms (total coliforms, E.coli) and inorganic tracers of anthropogenic waste (NO3−, SO42−, Cl/Br) from various sources: boreholes (12–76 m depth; n = 90), protected springs (n = 11) and municipal taps (n = 4). Our results show that NO3− and SO42− were elevated in groundwater sources in the Gulu city urban area and the Cl/Br ratio was elevated in springs, compared to concentrations in the more rural Aswa and Omoro County area (p < 0.05). Interestingly, human and animal waste indicators E.coli and Tryp:FA (the ratio of tryptophan-like to fulvic-like fluorescence) displayed no significant difference between rural and urban settings (p > 0.05), though total coliforms were significantly higher in rural boreholes (p < 0.05). The presence of a pollution source, pollution carrier and a breakdown of a sanitary barrier at the borehole, as spot-checked by a visual sanitary risk assessment, was significantly associated with groundwater E.coli abundances. Evidence suggests monitoring and mitigation should be improved for all water types in Gulu District to meet WHO and Uganda Standard guidelines for potable water. This study offers valuable insights for water management planning and risk assessment of community water sources particularly in the context of East Africa and similar settings.