Microplastic (MP) pollution is a global environmental issue, and traditionally treated wastewater has been identified as a source of land-based microplastics into the aquatic environment. This study evaluated the performance of a pilot-scale biofilter to polish wastewater treatment plant (WWTP) effluent before it enters the environment. The filter was divided into four zones, allowing the concentration of microplastics to be followed through the filter. It was fed with secondary effluent from a conventional WWTP in Denmark. The raw effluent from the WWTP contained 917 items m−3 which corresponded to a mass concentration of 24.8 μg m−3. After the top layer of the biofilter, the concentration had decreased to a median value of 197 item m−3 and 2.8 μg m−3, indicating an overall removal efficiency of 79% in terms of particle number and 89% in terms of particle mass. We also observed a tendency that MP of larger size and higher particle mass were more likely to be retained. After the last filtration zone, all MP larger than 100 μm had been removed. The results of this study demonstrate that biofilters are able to lower the MP abundance in treated wastewater significantly, but a complete removal is not ensured, hence some MP, particularly small-sized ones, can still be discharged into the receiving environment.