Wet wipe pollution is increasing in aquatic ecosystems. In urban catchments, wipes can become dominant plastic substrata for microbial colonization. It has been described that plastic can be colonized by pathogens and antibiotic‑resistant bacteria, with growing evidence that plastic surfaces may also facilitate their occurrence, representing potential health risk spots. Effluents from wastewater treatment plants are known to be sources of several antibiotic-resistant pathogenic bacteria from the ESKAPEE group. We hypothesize that wet wipes may provide novel substrata in aquatic ecosystems that facilitate the microbial establishment given their fibrous plastic structure. The combination of a high organic matter retention capacity and the hydrophobicity of plastic fibers may additionally promote denser or compositionally distinct biofilm assemblages. These features may enhance the potential for wet wipes to act as physical supports for pathogenic cell lines. To address this hypothesis, a biofilm colonization experiment was conducted in a flume mesocosm facility and in three urban rivers impacted by wastewater treatment plant effluents, all located near Barcelona (Spain). We compared the 4‑week colonization of two wet wipes with different plastic compositions to that of two selected inorganic and organic control substrata. DNA extractions of biofilms on the different substrata were performed, and targeted DNA qPCR assays for five pathogens (Aeromonas caviae, Clostridioides difficile, Clostridium perfringens, Klebsiella pneumoniae, and Pseudomonas aeruginosa) and four antibiotic resistance genes (tetA, blaCTX-M, sul1, and ermB) were conducted. To contextualize these results, occurrences of the main functional microbial groups within such periphytic-like assemblages were assessed by qPCR assays targeting phototrophs, microeukaryotes, and fungi. The metabolic activity (respiration rates) of the biofilms was also performed. Preliminary results show a high variability among stream sites and experimental substrata in the relative abundances of pathogen DNA targets. Statistical analyses of these data are currently underway to examine the drivers of this variability and elucidate whether biofilms in wipes hold pathogenically distinct microbial communities. These results will be shown in the presentation.