Microplastics (MPs) are a large group of plastic particles (1–1000 µm) and have been found literary everywhere around the world. When MPs enter the aquatic environment, different inorganic and organic substances start to adsorbed on the MPs surface, followed by the colonization of microorganisms. For biofilm characterization, the most popular methods are surface characterization by microscope and microbial community structure determination. However, many more methods can help characterize biofilm, but they have not been often used. In our study, polyethylene MPs (confirmed by FTIR), extracted from a facial scrub, were incubated in freshwater (Glinscica River) for 15 weeks (23 ± 2 °C, 125 rpm, light/dark 16h/8h). Every week, MPs were filtered and added to a new freshwater in order to ensure enough nutrients and microorganisms. The most visible and stable biofilm occurred after several weeks of incubation. Therefore, we evaluated changes in biofilm composition from week 13 to week 15. Within two weeks, the amount of biofilm on microplastics increased from 34 ± 8% to 43 ± 6%. An integral part of each biofilm are also extracellular polymer substances (EPS), which increased from 0.29 ± 0.05 mg/gbiofilm to 0.59 ± 0.10 mg/gbiofilm. Chlorophyll a content was 0.18 ± 0.03 mg/gbiofilm and 0.37 ± 0.11 mg/gbiofilm at week 13 and 15, respectively, which indicated that microalgae are likely to be present in the biofilm. Ureases are indispensable enzymes in numerous organisms, therefore the determination of urease activity indicate the activity of microorganisms in biofilm. Urease activity increased from 0.28 ± 0.01 mghydrolysed N/gbiofilm to 0.95 ± 0.27 mghydrolysed N/gbiofilm within two weeks. We also observed changes of MPs behaviour, the density of particles significantly increased with time, and they started to sink.