In recent years, the demand for reverse osmosis (RO) processes to reuse treated wastewater has been growing, and this has inevitably led to an increase in RO concentrate (ROC). The aim of this study was to assess the effect of total dissolved solids (TDS) in ROC on microbial activity and diversity. Oxygen uptake rates and pyrosequencing of bacterial 16S rRNA genes showed the following: with no TDS injected, stable NO3−N concentrations with an average of 2.34±0.96 mg/L were observed in the effluent; on injecting TDS, the mean NO3−N concentration was 4.22±2.96 mg/L in the effluent and microbial communities were more diverse. This is in contrast with the state of the effluent treated conventionally. These findings suggest that TDS injection may (1) diversify microbial species, (2) reduce relative abundance of certain microbial species involved in removing high concentrations of nitrogen and organic matter, and (3) decrease microbial activity and denitrification efficiency.