A large-scale field experiment was used to investigate the long-term effects of a single application of manure or inorganic fertiliser on microbial communities in the topsoil and subsoil of a cropping field in south-west Victoria. Poultry litter (20 t ha–1) and fertiliser (with equivalent total nutrients to the manure) was either surface broadcast or deep ripped into the subsoil before sowing in 2014. Soil samples were collected from the 0–10 and 25–40cm horizons in each treatment immediately after harvest of the third successive crop in January 2017. Next-generation sequencing of the 16S and ITS rRNA genes was used to characterise the bacterial and fungal communities in the soil. Amendment type and method of placement had a limited effect on soil microbial community structure and diversity, three years after treatments were applied. Fungal communities exhibited weak responses to the poultry litter and fertiliser in comparison to a nil control, but none of the treatments had any detectable effect on bacterial communities. Differences in structure and diversity of microbial communities were overwhelmingly due to their vertical distribution in the soil profile, and not the application of different amendments to the soil by deep ripping or surface broadcasting. The strength and timing of the soil disturbance, plant selection effects and farm management history likely contributed to the lack of measurable response in the soil microbial community.