Background Arsenic is one of the major toxic elements in the environment and is also known to be carcinogenic, with several other health side effects in human beings. Due to the lack of a sufficient supply of treated piped water across Uganda, boreholes are increasingly becoming an important water source for both domestic and commercial purposes. We determined the concentrations of arsenic in water obtained from underground wells and biological samples from residents in the Amuria district of Uganda. Methods We conducted a field-based, cross-sectional observational study to determine arsenic levels in underground water sources in Amuria District. Water samples were collected from boreholes and underground wells, alongside biological samples, including hair and nails, obtained from residents. Arsenic concentrations were quantified using an Agilent Atomic Absorption Spectrometer. Results The arsenic levels in the borehole-water from the different villages were all extremely above the WHO maximum acceptable concentration of 10 μg/L. The arsenic concentration in the sampled borehole water ranged from 4040 ± 973.6 μg/L to 5767.14 ± 258.3 μg/L. The arsenic concentrations in the hair and nail samples collected ranged from 0.02 ± 0.008 μg/g to 0.0045 ± 0.002 μg/g and from 0034 ± 0.001 μg/g to 0.00084 ± 0.0004 μg/g, respectively. Conclusion The borehole-water samples collected from villages of Amuria district had a significantly higher arsenic concentration than the recommended (10 μg/L) by WHO. The arsenic was also present in the biological samples of the residents who rely on the borehole-water for domestic purposes, but no significant correlation was established between water and nail or hair arsenic levels.