A field method called gas push–pull test (GPPT) was previously developed for in-situ quantification of methane (CH4) oxidation by soil microorganisms. We examined whether natural-abundance stable carbon-isotope analysis of CH4, a common approach used to measure in-situ bioconversions, could be used as a quantitative tool to complement the GPPT method. During GPPTs strong isotope fractionation of CH4 due to molecular diffusion can occur. This effect was observed in laboratory experiments regardless of the GPPTs’ advective component (i.e., for different injection/extraction rates). Numerical simulations showed that if a GPPT is applied in soils with low microbial CH4 oxidation activities, isotope fractionation may be dominated by molecular diffusion rather than by consumption. Because diffusional and microbial isotope fractionation of CH4 occur simultaneously during a GPPT, CH4 isotope data alone from a single GPPT cannot be used to assess the bioconversion process. However, microbial fractionation may be estimated if the extent of diffusional fractionation is known. This can be achieved either by conducting two sequential GPPTs, with microbial activity being inhibited in the second test, or by estimating physical transport processes via co-injected tracers’ gas analysis. We present a case study, in which we re-analyzed GPPTs previously performed in the unsaturated zone above a petroleum-contaminated aquifer. At this field site the combination of sequential GPPTs, stable carbon-isotope analysis of CH4, and a modeling approach, which considers diffusion and microbial CH4 oxidation, was a powerful tool to estimate in situ both apparent Michaelis–Menten kinetic constants and the microbial kinetic isotope effect.