The cycling of N in soil is supported both directly and indirectly by numerous microbial processes. These processes affect ecosystem fertility, but can also generate forms of N which have detrimental environmental impacts, such as N₂O. Understanding drivers of biological communities involved in key N-transformations is therefore of much interest. The effects of physicochemical and environmental properties on the relative size (abundance within total DNA pool) of biological communities involved in 3 key N transformations were investigated. Soils from 14 locations spanning a rainfall gradient across 3 agricultural regions (Clare, Mallee, Balaclava) were sampled, with samples taken from the surface and at depth from each site. Based on PCA of physicochemical and environmental properties, the soils fell into 2 distinct groupings: Clare and Mallee + Balaclava ‘types’. The abundance of functional genes involved in N₂ fixation (nifH), ammonia oxidation (amoA), and nitrate reduction (narG) was quantified in DNA extracted from the soils using real-time PCR. The abundance of the nifH gene varied significantly with site (P = 0.03) but not depth, and no regional association with nifH gene abundance was found. Multivariate analysis indicated that the abundance of nifH was positively correlated with soil total C (ρ = 0.382; P = 0.006). Similarly, the abundance of narG varied with site (P < 0.001) and not soil depth. The abundance of narG was positively correlated with increasing rainfall (ρ = 0.417; P = 0.002). The abundance of amoA did not significantly vary between soils, but significantly decreased with soil depth (P = 0.006). The abundance of amoA was negatively correlated with soil electrical conductivity and positively with organic C (combined ρ = 0.44; P = 0.003). Whereas there was no relationship between the abundance of nifH and amoA or narG, the abundance of amoA was positively correlated with the abundance of narG (P < 0.001). These results indicate that the abundance of the N cycling genes is independently affected by different physicochemical or environmental properties. The interactions between soil, environment, and the functionally significant biological communities they support are complex. To gain fuller understanding of soil N cycling, the ecology of the various biological components affecting N-transformations must be investigated simultaneously.