<p>The selection of an appropriate fertilizer type is crucial for enhancing nitrogen utilization in crops and mitigating agricultural non-point source pollution. Research has demonstrated that organic fertilizers can significantly improve crop yields and enhance soil fertility by supplying a balanced array of nutrients, thereby addressing the pressing need to recycle agricultural waste. Recently, biochar-based fertilizers have garnered considerable attention due to their potential to reduce greenhouse gas emissions by sequestering carbon in biochar. Both organic and biochar-based fertilizers are recognized as environmentally sustainable options worthy of broader adoption. However, the question remains as to which of these fertilizers is more strongly recommended for agricultural practitioners. A six-month monitoring study was carried out in a long-term field trial to investigate the effects of different treatments, including chemical fertilizer, biochar-based fertilizer, organic fertilizer as a substitute for 50% of chemical fertilizer and a control group, on rice yield, nitrogen use efficiency, soil physicochemical properties, nitrogen losses, duckweed biomass, soil microbial functional genes and community. The results indicated that organic fertilizer significantly increased rice yield and nitrogen use efficiency, while reducing ammonia volatilization and nitrogen runoff losses, attributed to the higher plant uptake by rice and duckweed during the growing season. Additionally, higher <i>α</i>-diversity of bacterial communities was observed under organic fertilizer treatment. Biochar-based fertilizer promoted the transformation of the soil N cycle into the pathway for efficient plant N use but had limited improvement in rice production and pollution mitigation owing to the high pH of biochar, leading to more ammonia volatilization losses. Therefore, organic fertilizer as a substitute for chemical fertilizer is optimal fertilization for rice cultivation.</p>