During the scientific expedition GAZCOGNE2 at the Bay of Biscay nine gas seeps were sampled for the first time and their flux was measured using an in situ pressure-preservation sampler (PEGAZ, ©IFREMER). Overall, three sites were investigated to determine the nature and the origin of the gases bubbling at the seafloor and forming acoustic plumes into the water column, as this was the question raised fromthe first geologic study of the area.This has guided our study and accordingly corresponds to themain purpose of the present article.Thus, themolecular and isotopic (𝛿D and 𝛿13C) analyses revealed that the gas seeps were primarily composed of methane. Both methane and ethane are of microbial origin, and the former has been generated by microbial reduction of carbon dioxide. Heavier hydrocarbons accounted for less than 0.06% mol of the total amount. Despite the microbial origin of methane, the samples exhibit subtle differences with respect to the 𝛿13CCH4 values, which varied between −72.7 and −66.1‰. It has been suggested that such a discrepancy was predominantly governed by the occurrence of anaerobic methane oxidation. The PEGAZ sampler also enabled us to estimate the local gas fluxes fromthe sampled streams. The resulting values are extremely heterogeneous between seeps, ranging from 35 to 368mLn⋅min−1. Assuming a steady discharge, the mean calculated methane emission for the nine seeps is of 38 kmol⋅yr−1. Considering the extent of the seep area, this very local estimate suggests that the Aquitaine Shelf is a very appropriate place to study methane discharge and its fate on continental shelves.