Environmental DNA (eDNA) is a cost-efficient, non-invasive method to monitor fish populations, but the quantitative aspect of this technique (e.g., estimating biomass or densities) remains underexplored. Few studies have established relationships between fish DNA concentration and biomass/density. Here, we investigate the relationship between eDNA concentration (copies per liter) and trout biomass and densities estimated by electrofishing in mountain streams of Picos de Europa National Park (Spain). We assessed eDNA effectiveness in inferring biomass/density using 18S rRNA (18S) and cytochrome c oxidase I (COI) metabarcoding, and quantitative PCR with a COI-specific Salmo trutta primer, each performed with different datasets from the same sampling points. Salmonidae eDNA concentration positively correlates with trout biomass and density. Both 18S and specific-COI markers showed a significant increase in DNA concentration as trout biomass and density rose in electrofishing surveys. However, general COI did not exhibit significant trout DNA concentration and biomass/density relationships, despite providing greater taxonomic resolution at the species level. Further analysis exploring eDNA concentration and biomass/densities across different trout size classes (fry, juvenile, and adult) revealed that juvenile trout biomass contributed the most to the observed eDNA concentration – biomass/density relationship. Our results suggest that DNA concentration estimated from metabarcoding, when using an appropriately selected primer, can reliably indicate trout biomass and density in these mountain streams where trout is the dominant species. Although quantitative PCR showed similar trends, it had lower explanatory power. This study highlights the importance of integrating a quantitative framework in metabarcoding for ecological monitoring and biodiversity assessments. Factors such as amplicon length, genetic region, marker specificity, or fish size class can influence the relationship between sequencing reads and electrofishing data. This methodology could aid the conservation and management of fish populations and other communities, though further research is needed to extend these results and assess eDNA detection reliability.