Tillage and nitrogen (N) fertilization have been shown to affect soil abiotic and biotic properties, but it is unclear how these impact long-term tillage systems in subtropical climates. We studied a 48-y wheat cropping trial in semi-arid, subtropical Australia that applied different tillage practices (no-till [NT] and conventional till [CT]) and N fertilization rates (0 and 90 kg N ha-1, 0 N and 90 N, respectively) in a Vertisol from 0 to 10 cm soil depth. Total organic carbon (TOC) significantly increased only when N fertilizer was applied under NT. Total nitrogen (TN) concentrations increased when N fertilizer was applied but were unaffected by tillage. N-acetyl-β-D-glucosaminidase (NAG) activity was highest when N was applied and α-glucosidase (α-Glu) activity was lowest in the NT and 0 N treatments. Activities of β-glucosidase, phenol oxidase and phosphatase were unaffected by the management practices. While the bacterial community composition appeared most distinct in NT and 0 N conditions, fungal community composition, and bacterial and fungal alpha diversity remained unaffected by the treatments. Although wheat yield was only significantly influenced by N fertilization rates, both NT and N fertilization may be preferable for management as these soils had the most favorable TOC, TN, and enzymatic activity. These differences seen between treatments here are less pronounced than reported elsewhere, with this likely a result of the high clay content of the soil which can have a buffering capacity, or that these treatments have less of an impact over longer time scales. Nevertheless, we conclude that no-tillage and N fertilization remain important in influencing soil properties and functioning over decades in this Vertisol in a semi-arid subtropical region.