Since microplastics were recognized as a global environmental problem in the early 2000s, research began on possible solutions such as the removal of microplastics from waters. A novel and promising approach for this purpose is microplastics agglomeration-fixation using organosilanes. In this study, it is investigated how biofilm coverage of microplastics affects this process. The biofilm was grown on the microplastics by cultivating it for one week in a packed bed column operated with biologically treated municipal wastewater enriched with glucose. The biofilm was characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and Fourier-Transform infrared spectroscopy (FT-IR). The results show a partial coverage of the microplastics with attached bacteria and extracellular polymeric substances (EPS) after 7 days of incubation. Comparing five polymer types (polyethylene, polypropylene, polyamide, polyester, and polyvinyl chloride) and three organosilanes, the biofilm coverage caused a reduced removal efficiency for all combinations tested as it changes the surface chemistry of the microplastics and therefore the interaction with the organosilanes tested in this study. Treatment of biofilm covered microplastic with ultrasound partly recovers the removal. However, the results underline the importance of simulated environmental exposure when performing experiments for microplastic removal.