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• Emerging contaminants detected in the rural water supply systems of Boyacá, Colombia. • Caffeine (>5 µg/L) and coliforms were present due to poor treatment practices. • Twenty-two ARGs persisted post-treatment, likely from runoff and biofilm formation. • The results emphasize the need for improved water treatment and protection of water sources. Access to safe drinking water remains a significant challenge in developing countries, where water sources (WS) often serve as hotspots for emerging contaminants (ECs) that include antibiotic resistance genes (ARGs), posing increasing public health risks. In Boyacá, Colombia, approximately one third of the rural population lacks access to safe drinking water. This study evaluated ECs in six rural water supply systems previously identified with sanitary risks. Between 2023 and 2024, a total of 54 samples were collected from WS, treatment plant effluents, and distribution networks (DN) during the high and low rainfall seasons. Physicochemical and microbiological parameters, mercury, organochlorine pesticides (OCPs), and ECs were analyzed using spectrophotometric, chromatographic, and molecular techniques. The results showed operational deficiencies, particularly empirical coagulant dosing, leading to elevated turbidity (2.44 ± 2.20 NTU) and the presence of coliforms and Escherichia coli in 80% of the samples. Although most of the physicochemical parameters met Colombian regulations, caffeine (>5.0 µg/L) was detected. Furthermore, 22 ARGs were identified, with low removal efficiency (31%) during treatment and resurgence in DN. In particular, the GES (Guiana Extended-Spectrum β-lactamse ) gene, linked to broad-spectrum β-lactam resistance, persisted throughout the treatment processes. The presence of these genes is associated with agricultural runoff at intake points and the formation of biofilms within distribution systems. These findings emphasize the urgent need to protect WS and improve infrastructure and operational practices. Future studies are recommended to analyse the temporal dynamics of ARGs and assess the effectiveness of treatment technologies to reduce health risks in rural communities.