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The quality of water supply systems is still a major problem in developing countries, especially in rural areas. The acute bioluminescence V. fischeri inhibition assay is widely recognized as a toxicological method that can be used to detect the acute effects of different contaminants. In this study, the physicochemical characteristics and toxicology of 72 water samples collected in 18 rural aqueducts located in Boyacá (Colombia) were evaluated. The primary economic activities identified as potential influencers of water quality in the water supply basins were agriculture (n = 3), livestock (n = 2), and domestic sewage discharge (n = 1). The average luminescence inhibition rate was 66%, with a minimum of 29%, and a maximum of 97%. A total of 85% of the tested samples (n = 61) had “moderate acute hazard”, while 15% (n = 15) had “acute hazard”. A total of 95% of the aqueducts distributed water with high risk. There was a weak positive correlation between the apparent color and the V. fischeri inhibition rate (p < 0.05). The water treatments, including disinfection, and the economic activities had no correlation with the inhibition rate of luminescent bacteria. The results of this investigation can be used by sanitary authorities to incorporate future toxicological monitoring of chemical contaminants, such as humic substances and metals, into water-quality monitoring in rural areas.