The southern area of China, particularly Guangxi region, contributes significantly to the nation’s sugar industry. Nevertheless, this region faces challenges due to excessive use of nitrogen fertilizer in sugarcane cultivation, leading to land degradation. Biofertilizers (BF) signifies an important turning point in this challenging scenario globally. However, there is few literature on the co-application of biofertilizers (BF) and nitrogen (N) fertilizers and their effect on sugarcane rhizosphere microbial community, soil chemical properties, and above-ground growth. This study employed the combination of six different treatment in a netted greenhouse experiment and the results showed a significant variations in sugarcane biomass, soil chemical properties and rhizosphere bacterial community. Further, in comparison to the control and only N fertilization, the combined application of biofertilizer and nitrogen (BF+N) significantly affected the rhizosphere microbes as per the Shannon and Chao1 index (p < 0.05). The altered relative abundance of microbes significantly affected soil chemical properties, including pH, total carbon, total nitrogen, ratio of carbon and nitrogen, ammonium nitrogen, and nitrate-nitrogen. Ternary plot analysis further showed the enriched bacterial genera i.e., Burkholderia, Sphingomonas, Meosrhizobium, Rhizobacter, Nitrospirae, and Dyella, as a result of BF amendment. Redundancy analysis also validated the results and showed that soil organic matter, total carbon, and its ratio to nitrogen and ammonium nitrate were the key edaphic factors in altering the microbial community of sugarcane rhizosphere. In conclusion biofertilizer (1500 kg.ha⁻¹) combined with 150 kg.ha⁻¹ of urea (BF2) could be an effective strategy for enhancing sugarcane growth, reducing chemical fertilizers, and moving towards sustainable soil environment in continuous sugarcane cropping system.