Silver carp (Hypophthalmichthys nobilis), bighead carp (H. molitrix) and their hybrids, collectively known as bigheaded carps, have been introduced to Lake Balaton, Hungary. The current stock sizes are difficult to assess. We investigated environmental DNA (eDNA) techniques targeted for bigheaded carps, assessed the spatial distribution of eDNA in Lake Balaton, compared eDNA concentrations to environmental variables to assess potential habitat selection based on those variables, and provided an estimate of biomass of bigheaded carps relative to eDNA shedding rates per unit biomass observed in controlled experiments. Water samples were collected from 70 sites in an array across the lake. Biomass estimation was calculated using mean eDNA concentration obtained by quantitative PCR of the samples and previously determined eDNA shedding rates of bigheaded carps under controlled conditions in a laboratory. Concentration of eDNA was highly variable between sites, resulting in wide confidence intervals. Basins did not significantly differ in eDNA concentration, and there were no strong relationships between environmental variables and eDNA concentration, indications that bigheaded carps use the entire lake. The model provided an estimate of 4,830 metric tonnes (2,750–8,030 tonnes) of bigheaded carps in Lake Balaton, or 81.0 kg/ha. The eDNA method produced a value close to previous estimates by traditional means of total biomass of bigheaded carps in the lake, and like traditional methods, there was a broad confidence interval on the estimate of the mean. The results of the present study support the utility of aquatic eDNA analysis, and the need for further comparisons with fisheries methods and supporting data from laboratory studies.