Diazotrophic bacteria potentially supply substantial amounts of biologically fixed nitrogen to crops, but their occurrence may be suppressed by high nitrogen fertiliser application. Here, we explored the impact of high nitrogen fertiliser rates on the presence of diazotrophs in field-grown sugarcane with industry-standard or reduced nitrogen fertiliser application. Despite large differences in soil microbial communities between test sites, a core sugarcane root microbiome was identified. The sugarcane root enriched core taxa overlap with those of Arabidopsis thaliana raising the possibility that certain bacterial families have had long association with plants. Reduced nitrogen fertiliser application did not increase the relative abundance of root-associated diazotrophs or nif gene counts. Correspondingly, low nitrogen fertiliser crops had low biomass and nitrogen content reflecting a lack of major input of biologically fixed nitrogen, indicating that manipulating nitrogen fertiliser rates does not improve sugarcane yields by enriching diazotrophic populations under the test conditions. Standard nitrogen fertiliser crops had improved biomass and nitrogen content, and corresponding soils had higher abundances of nitrification and denitrification genes. These findings highlight that achieving a balance in maximising crop yields and minimising nutrient pollution associated with nitrogen fertiliser application requires understanding of how microbial communities responds to fertiliser use.