Identifying the source of methane (CH4) in groundwater is often complicated due to various production, degradation and migration pathways, particularly in settings where there are multiple groundwater recharge pathways. This study demonstrates the ability to constrain the origin of CH4 within an alluvial aquifer that could be sourced from in situ microbiological production or underlying formations at depth. To characterise the hydrochemical and microbiological processes active within the alluvium, previously reported hydrochemical data (major ion chemistry and isotopic tracers (3H, 14C, 36Cl)) were interpreted in the context of CH4 and carbon dioxide (CO2) isotopic chemistry, and the microbial community composition in the groundwater. The rate of observed oxidation of CH4 within the aquifer was then characterised using a Rayleigh fractionation model. The stratification of the hydrochemical facies and microbiological community populations is interpreted to be a result of the gradational mixing of water from river leakage and floodwater recharge with water from basal artesian inflow. Within the aquifer there is a low abundance of methanogenic archaea indicating that there is limited biological potential for microbial CH4 production. Our results show that the resulting interconnection between hydrochemistry and microbial community composition affects the occurrence and oxidation of CH4 within the alluvial aquifer, constraining the source of CH4 in the groundwater to the geological formations beneath the alluvium.