Aquaculture wastewaters have been used in rice irrigation directly or indirectly. Antibiotics in aquaculture wastewater entering the rice fields with irrigation will affect the soil physicochemical properties, microbial properties, and nitrogen conversion. A pot experiment irrigated with simulated aquaculture wastewater was performed to study the effects of sulfamethazine in aquaculture wastewater on nitrogen concentration and conversion-related microorganisms in rice fields with different irrigation modes. Sulfamethazine (500 ng/L, 1500 ng/L, and 3000 ng/L) decreased the NH4+ concentration at the late tillering stage and NO3− concentration at the late tillering and jointing–booting stages (p < 0.05) but increased the NH4+ concentration at the late tillering stage (p < 0.05). Sulfamethazine (3000 ng/L) promoted the lowest nitrogen conversion gene (amoA, nirS, and nirK) abundances and the most special community structure of nitrogen conversion microorganism under mild alternate wetting and drying (AWD). Furthermore, Nitrosospira_sp._KAN8, belonging to ammonia-oxidizing bacteria (AOB), was sensitive to sulfamethazine. Flora with the same nitrogen conversion genes exhibited different variations under the same treatment. The results show that antibiotic and mild AWD caused more serious adverse effects to soil nitrogen conversion and nitrogen conversion microorganisms, which will increase the environmental risks of sulfamethazine. It can provide a basis for the scientific and rational use of aquaculture water to irrigate rice fields.