Adding herbicides to sewer lines, a common practice for controlling root intrusion in sewer pipes, may adversely impact downstream wastewater treatment by inhibiting nitrification and denitrification performance. This study investigated the effects of herbicides, namely diquat, triclopyr, and 2-methyl-4-chlorophenoxyacetic acid (MCPA)-dicamba, on these processes. Various parameters were monitored, including oxygen uptake rate (OUR), nutrients (NH3–N, TP, NO3–N, and NO2–N), chemical oxygen demand (COD), and herbicide concentrations. It was found that nitrification was not affected by OUR in the presence of each herbicide at various concentrations (1, 10, and 100 mg L−1). Additionally, MCPA-dicamba at various concentrations demonstrated minimal inhibition in the nitrification process compared to diquat and triclopyr. COD consumption was not affected by the presence of these herbicides. However, triclopyr significantly inhibited NO3–N formation in the denitrification process at various concentrations. Similar to nitrification process, both COD consumption and herbicide reduction concentration were not affected by the presence of herbicides during the denitrification process. Adenosine triphosphate measurements showed minimal impact on nitrification and denitrification processes when herbicides were present in the solution up to a concentration of 10 mg L−1. Tree root kill efficiency experiments were performed on Acacia melanoxylon. Considering the performance on nitrification and denitrification process, diquat emerged as the best herbicide option (concentration of 10 mg L−1), with a 91.24% root kill efficiency.