The 'One Health' approach to CKDu research has not yet reported the impact of drinking water exposure on bovine kidney functions in CKDu endemic areas. This study investigated the associations of fluoride and trace elements in high-hardness drinking water with DOM components on bovine kidney functions in CKDu endemic areas. Water samples (n = 190) were collected from drinking water sources in both CKDu-endemic and non-endemic regions, and blood samples were collected from 162 milking cows. Pearson correlogram showed strong positive correlations within the cluster of drinking water F-, Hardness, Ca2+, Mg2+, Sr2+ with the organic components of HIX, C1, SUVA, UVAbs254 and TOC in CKDu endemic areas. In cow serum biochemistry parameters, it was found that Blood Urea Nitrogen (BUN), Serum Creatinine (SCR) and Total Protein (TP) values were significantly higher (p < 0.01) in CKDu endemic area group (T) than those in the control group (C). Serum Albumin (SAL) levels were significantly lower (p < 0.01) in the T group, suggesting possible chronic renal stress. The T group showed significant positive correlations (p < 0.01) with F-, Na+, and Hardness levels for SCR and TP, except for Hardness with BUN. Nevertheless, SAL exhibited significant (p < 0.05) negative correlations with Hardness, Na+ and F- within the T group. Thus, the study identified exploratory, association-based thresholds for selected drinking water and serum biomarkers to support screening of higher-risk cows. Given the observational design, these findings indicate potential preventive screening values within a One Health framework. However, independent validation across diverse geographic and epidemiological settings is recommended.