Abstract In recent years, the analysis of environmental DNA (eDNA) has significantly improved, allowing for high‐resolution species identification and possible biomass quantification from water samples. Fisheries management typically requires monitoring of catches, including precise information about bycatch quantities to make sound assessments of exploitation rates. Bycatch assessment is particularly challenging in large catches (>500 T), and the current practice of visual assessment of subsampled catches is time‐consuming, requires extensive labor, and often has low precision. We explored the feasibility for applying eDNA‐based methods for studying catch composition using the pelagic North Sea herring fishery with bycatch of mackerel as a case. First, we experimentally simulate a series of catches using a range of herring and mackerel weight proportions to establish relationships under real fisheries scenarios. The relationship is subsequently used to estimate the biomass of mackerel bycatch from eDNA from three herring catches, by sampling and comparing processing water both onboard ships and at the processing factory. All samples are analyzed using species‐specific quantitative PCR (qPCR). The experiments reveled a strong correlation between DNA and weight fractions characterized by a constant overrepresentation of mackerel DNA compared to expected mackerel weight. We found that eDNA‐based and visual methods applied to the same landing reflect the within catch variability in species composition alike, however, the methods can show disparity in total estimates of mackerel biomass. Accounting for haul mixing within total landed catches increases the precision of the factory and ship eDNA‐based estimates for the same catch. We show that eDNA‐based bycatch estimates provide coherent quantitative data, and likely improve quality and reduce costs of collecting fisheries‐dependent data and thereby contribute to securing sustainable fisheries.