Nature-based wastewater treatments are an economic and sustainable alternative to intensive technologies in rural areas, although their efficiency needs to be improved. This study explores technological co-operation between zooplankton (e.g., Daphnia magna) and bacterial and algal biofilms in a 1.5 m3 zooplankton-based reactor for the on-site treatment of secondary urban wastewater. The efficiency of the reactor was evaluated over a 14-month period without any maintenance. The results suggest a low seasonality effect on nutrient polishing (organic matter and nitrogen) and the removal of solids (TSS and turbidity). The best performance, involving a decrease in organic carbon, nitrogen, E. coli loads, and solid content was achieved in winter when operating the reactor at 750 L d−1. Under these conditions, the quality of the effluent water was suitable for its reuse for six different purposes in conformance with Spanish legislation. These results demonstrate that the zooplankton-based reactor presented here can be used as an eco-sustainable tertiary treatment to provide water suitable for reuse. On-site research revealed that the robustness of the reactor against temperature and oxygen fluctuations needs to be improved to ensure good performance throughout the year.