Natural gas seeps in the Alpine region are poorly investigated. However, they can provide useful information regarding the hydrocarbon potential of sedimentary Alpine units and related geofluid migration, typically controlled by pressurized gas accumulations and tectonics. A gas seep located near Giswil, in the Swiss Northern Alps, was investigated, for the first time, for molecular and isotopic gas composition, methane flux to the atmosphere, and gas flux variations over time. The analyses indicated that the gas was thermogenic (CH4 > 96%; δ13C1: −35.5‰ to −40.2‰) and showed evidence of subsurface petroleum biodegradation (13C-enriched CO2, and very low C3+ concentrations). The source rock in the region is marine Type II kerogen, which is likely the same as that providing thermogenic gas in the nearby Wilen shallow well, close to Lake Sarnen. However, the lack of δ13CCO2 and δ13C3 data for that well prevented us from determining whether the Wilen and Giswil seeps are fed by the same reservoir and seepage system. Gas fluxes from the Giswil seep, measured using a closed-chamber system, were significant and mainly from two major vents. However, a substantial gas exhalation from the soil occurs diffusely in an area of at least 115 m2, leading to a total CH4 output conservatively estimated to be at least 16 tonnes per year. Gas flux variations, monitored over a 1-month period by a special tent and flowmeter, showed not only daily meteorological oscillations, but also an intrinsic ‘pulsation’ with periods of enhanced flux that lasted 2–6 h each, occurring every few days. The pulses are likely related to episodes of gas pressure build-up and discharge along the seepage system. However, to date, no relationship to seismicity in the active Sarnen strike-slip fault system has been established.