Soil water repellency (SWR) is an agricultural concern as it limits plant available water, leading to decreased crop yields at times when a drying climate and expanding population place increasing pressure on food production. Here we assessed a microbial inoculation that secretes biosurfactants and lipolytic enzymes and a chemical surfactant's ability to mitigate SWR. We applied either Bacillus subtilis (Gm), surfactant (Gs), Bacillus subtilis with surfactant (Gms) or no treatment (G) to a gamma-irradiated, naturally water-repellent, agricultural soil, with the same soil as an un-sterilised, untreated control (C). Soil water content and the persistence of SWR in a water-repellent core (4 × 4 × 3 cm) were measured during 19 days of repeated soil wetting and drying. Meso-scale, 3D electrical resistivity tomography (ERT) was developed for the continuous, non-destructive measurement of the patterns and volume of water content in the soil, however, effectiveness was limited as the electrical current (5 mA) restricted microbial activity. Destructive measurements of soil water content and potential SWR were conducted in 1 cm soil layers throughout each core. The persistence of SWR decreased over time in all treatments apart from treatment G. SWR was removed by day 12 within the Gm treatment, and was not exhibited immediately after wetting in treatments Gs and Gms. The Gs treatment also increased water content immediately while treatment Gm increased water content after eight days of incubation, with soil saturated by day 19 of the incubation. Persistence of SWR significantly varied with depth, with the highest persistence in the 1–2 cm soil layer. Our findings illustrate that in order to remove SWR, without the effect of chemical surfactants, first microbial degradation of the molecules attributing to SWR has to occur. This indicates a biological mechanism as the driver for non-reversible SWR breakdown rather than a solely physical process.