Nitrogen is an essential plant nutrient, but N fertilization contributes to greenhouse gas emissions through its production and application as well as nitrous oxide emissions when applied to soil. Diazotrophic bacteria, known to modify root architecture and increase nutrient uptake, have been proposed as a potential strategy to improve nitrogen use efficiency (NUE) in sugarcane cultivation. The objective of this study was to evaluate the efficiency of N use from ¹⁵N ammonium sulfate applied to different sugarcane varieties inoculated with diazotrophic bacteria. The experiment was conducted in pots filled with soil (100 kg pot⁻¹). The sugarcane varieties tested were RB867515 and RB92579. A commercial diazotrophic bacteria inoculant for sugarcane was used. The experimental design was a randomized block design with four replicates. The treatments were as follows: control without inoculation, inoculation with five strains of bacteria, with or without nitrogen fertilization. The evaluations were performed on different parts of the plant. At 360 days after transplanting, the accumulation of N, fresh mass, dry mass, and the proportion of excess ¹⁵N were determined. In the studied sugarcane varieties, the efficiency of ¹⁵N fertilizer use was 60%, with no influence from inoculation.