Rice-fish mutualistic production systems rationalise the use of water and soil resources in an improved approach to sustainable food production. However, drivers of fungi community structure in paddy soil, including effects of nitrogen (N) application rate, are unclear in these systems. Here, we assessed soil fungi community and soil physicochemical responses in paddy soil to contrasting rates of N application in a rice-fish system. To clarify the mutualistic effects, the rice-fish system was compared with a standard rice monoculture under a 325.5 kg ha−1 N application rate. The results showed that N application rate affected abundance of paddy soil fungi (P < 0.05). Alpha diversity and richness of fungi were lower in the rice-fish system, but evenness increased with a decrease in N application rate, while the rate of N determined diversity of soil fungi in the rice-fish system. Dominant genera in the two systems differed, and soil physicochemical properties were more important drivers of soil fungi community structure in the rice-fish mutualistic system than in rice monoculture. Total N, available N and P regulated the abundance of dominant fungi. Our results indicate that management of soil fungi may contribute to sustainable agricultural production.