Mercury contamination from artisanal and small-scale gold mining (ASGM) remains a major concern in riverine environments and exposed communities. This study applied a multi-matrix approach in an ASGM-impacted area of Bungo District, Jambi Province, Indonesia, integrating mercury analysis in water, sediment, fish muscle, and human hair with kidney-related biomarkers. Mercury was not detected in the analyzed water samples, whereas sediment mercury was detected at several sites, with mean concentrations ranging from 0.02 to 0.10 mg/kg. Mercury was detected in all fish samples, with mean concentrations ranging from 0.05 to 0.17 mg/kg. Among 50 residents, hair mercury ranged from 0.17 to 6.15 mg/kg, with a mean of 1.01 ± 0.98 mg/kg; 10.0% of participants had concentrations ≥2.0 mg/kg. The mean UACR was 16.31 ± 32.55 mg/g, the mean eGFR was 107.76 ± 40.62 mL/min/1.73 m², and 18.0% of participants had at least one altered kidney-related indicator. These findings indicate that water-only monitoring may underestimate mercury relevance in ASGM-affected river systems because mercury can be retained in sediments and detected in biological matrices even when surface-water mercury is below the analytical detection limit. The results support a multi-matrix monitoring approach using sediment, fish, and human biomarkers to improve interpretation of contamination persistence, bioaccumulation potential, and exposure significance. However, because the study was cross-sectional, based on total mercury analysis, and limited in sample size for fish and human health interpretation, the findings should be interpreted as evidence of a plausible environmental–food chain–human exposure continuum rather than proof of causality.