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The use of environmental DNA (eDNA) is a promising approach for the detection of aquatic species, including species at risk. One freshwater mussel species of interest in Atlantic Canada, the brook floater (Alasmidonta varicosa), is listed as being of Special Concern under the Species at Risk Act in Canada and as Vulnerable on the International Union for Conservation of Nature Red List. Further scientific data regarding species distribution and critical habitat is needed for the protection and conservation of this species. The aim of this study was to design, optimize, and apply a species‐specific quantitative polymerase chain reaction assay for the detection of brook floater from eDNA samples, and to assess temporal variability in brook floater eDNA quantities. Through an eDNA survey performed in New Brunswick rivers in 2017 and 2018, brook floater DNA was found at a total of 16 out of 56 sites sampled. The amount of brook floater DNA detected at all 16 sites was always below the theoretical limit of detection of the assay, and, as such, results were classified as either ‘inconclusive’ or ‘suspected’. The co‐detection of eastern pearlshell (Margaritifera margaritifera), a more abundant freshwater mussel species in Atlantic Canada, was successfully used as a natural positive control. Temporal variability in the amount of eDNA found in the water was also assessed at a site with a known brook floater population and minimal variability in eDNA quantities was observed from May to September. These results provide researchers and managers with a new tool for the detection of the brook floater in support of conservation and monitoring efforts.