Microplastics (plastic debris with diameter <5mm) are of particular concern to the environment. However, there is a scarcity of information concerning the effects of conditioning films on bacterial colonization of microplastics in freshwater ecosystems. The formation of conditioning films on substrate surfaces is a critical step in the priming of substrates for bacterial colonization in aquatic systems. Conditioning films are comprised of dissolved organic solutes that are deposited on to surfaces of substrates, which attract bacterial colonizers. Moreover, the thicknesses of conditioning films are influenced by the physicochemical properties of substrate surfaces. This study aimed to understand the effects of different conditioning films on bacterial colonization of microplastic surfaces in freshwater. Five types of conditioning films were analyzed: Bovine Serum Albumin (BSA), sodium alginate (medium and very low viscosity), humic and fulvic acid; all are components of biofilms on four types of microplastic disks (diameter <5mm): polypropylene (PP), polystyrene (PS), high-density polyethylene (HDPE), and low-density polyethylene (LDPE). The disks were analyzed for conditioning film thicknesses using AFM (atomic force microscopy) and 16S rRNA sequencing to determine compositions of bacterial communities in the presence of different conditioning films. Understanding these questions will provide insights on fates of microplastic debris in freshwater.