Heterotrophic nitrifying bacteria are attracting increasing interest due to their advantages in treatment of wastewater with high nitrogen concentration. In this work, four bacterial isolates (WS-1, WS-4, WS-6, and WS-7) with the capability of simultaneous heterotrophic nitrification and aerobic denitrification from soil, sludge, and water by a fast isolation procedure were studied. Isolates WS-1, WS-6, and WS-7 were identified as Pseudomonas sp. and WS-4 as Agrobacterium sp. based on 16S rRNA gene sequence analysis. All four isolates could utilize ammonia, nitrate, and nitrite as sole nitrogen source and the metabolic characteristics were shown to be related with the genus. In addition, Agrobacterium sp. WS-4 accumulated less nitrate than the three Pseudomonas sp. strains with ammonia as the sole nitrogen source. When nitrate or nitrite was the sole nitrogen source, Agrobacterium sp. did not produce ammonia, and it consumed the least nitrogen compared with the three Pseudomonas sp. isolates. Our studies implied that Pseudomonas sp. strains had great potential in treating high nitrogen in wastewater.