Novel heterotrophic bacterial strains-Bzr02 and Str21, effective in nitrogen transformation, were isolated from sequential sedimentation-biofiltration systems (SSBSs). Bzr02, identified as Citrobacter freundii, removed up to 99.0% of N-NH4 and 70.2% of N-NO3, while Str21, identified as Pseudomonas mandelii, removed up to 98.9% of N-NH4 and 87.7% of N-NO3. The key functional genes napA/narG and hao were detected for Bzr02, confirming its ability to reduce nitrate to nitrite and remove hydroxylamine. Str21 was detected with the genes narG, nirS, norB and nosZ, confirming its potential for complete denitrification process. Nitrogen total balance experiments determined that Bzr02 and Str21 incorporated nitrogen into cell biomass (up to 94.7% and 74.7%, respectively), suggesting that nitrogen assimilation was also an important process occurring simultaneously with denitrification. Based on these results, both strains are suitable candidates for improving nutrient removal efficiencies in nature-based solutions such as SSBSs.