The objective of this study was to determine how soil disturbance and soil water deficit alter colonisation of roots by naturally occurring arbuscular mycorrhizal (AM) fungi and rhizosphere bacteria. Soil cores were collected at the end of summer from a cropped paddock with a 5-year history of no-tillage in south-western Australia which has a Mediterranean climate. Well-watered and water-stressed treatments were maintained at 70 and 35% field capacity, respectively. AM fungal colonisation was determined using microscopy, rhizosphere bacterial community composition was assessed using barcoded PCR-amplified bacterial 16S rRNA genes, and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis of functional gene prediction was used to characterise the rhizosphere functionality for some nitrogen (N) cycling and soil carbon (C) degradation. Mycorrhizal colonisation and plant growth were reduced by disturbance under water stress but not for well-watered soil cores. The rhizosphere bacterial community composition shifted with both soil disturbance and soil moisture, and there was an interaction between them. Disturbance decreased the relative abundance of Proteobacteria and Acidobacteria and increased the relative abundance of Actinobacteria and Firmicutes in water-stressed soil. The predicted abundance of some genes involved in N reactions was negatively influenced by both disturbance and soil moisture, but the predicted abundance of C degrading predicted genes was only marginally affected. This study highlighted how soil disturbance prior to seeding can alter soil biological processes that influence plant growth and that this could be most pronounced when water is limiting.