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Iron biofouling of wells can significantly impact the performance of a groundwater extraction system. A subsurface drainage scheme (Wakool, Australia) designed to reduce waterlogging was used to identify some of the relationships between aquifer properties and well biofouling. Piezometers drilled radially one metre from two biofouled wells showed that during normal well operation the concentration of dissolved iron (Fe2+) entering the groundwater well was highly localised around the site and with depth. CCTV survey of the biofouling on the well screens supported these findings of localised iron concentrations. Dissolved oxygen (DO) measured during pumping and under non-pumping conditions (aquifer DO) showed that oxygen was not a limiting factor, whereas stalked bacteria (Gallionella sp.) were only found in the biofouled wells. The wellhead water therefore represents only a composite of all the waters entering the well and does not indicate the possibility of localised iron concentrations in a shallow aquifer. The degree of iron biofouling within a groundwater well is therefore related directly to the presence of dissolved iron in the groundwater, as well as various oxidative processes occurring as the groundwater enters the well screen and its subsequent extraction. The distribution of iron biofilms on the well screen reflects these processes; however, the presence of well biofouling cannot always be linked to a decrease in well screen performance, but can have an impact on the overall performance of the groundwater extraction system.