Nitrification and denitrification processes occur simultaneously in aerobic wastewater biofilms. Although wide regions of the world have average temperatures of less than 15 °C for a half year, few studies have investigated the nitrogen removal by nitrification and denitrification in a single-stage aerobic biofilm reactor used for treating real wastewater under low-temperature conditions. This study showed successful wastewater treatment in a high average nitrogen removal rate of 78% at low water temperatures by simultaneous nitrification and denitrification in a rotating biological contactor (RBC) biofilm. Batch operations using the RBC to evaluate the rates of ammonium decrease at low temperatures demonstrated that the rate of ammonium decrease at 8 °C was 76% of that at 20 °C. Daily monitoring of nitrification and denitrification rates suggested that the denitrification rate was highly correlated with the nitrification rate. Next-generation sequencing (NGS) analysis revealed the presence of diverse and abundant denitrifying bacteria and aerobic bacteria in the RBC biofilm more than those in the activated sludge samples, which probably enabled the achievement of the high nitrogen removal rates at such low temperatures. Furthermore, correlation with the colony counts showed that the NGS analysis had the quantification range of three orders of magnitude (from 0.001% to 1%).