Parabens, as emerging environmental contaminants, raise significant concerns due to their potential to disrupt microbial ecology and dynamics. Their widespread use and continuous discharge result in their widespread distribution and accumulation in aquatic environments due to their incomplete removal by traditional wastewater treatment processes. Bacterial biofilms in these water systems are continually exposed to parabens, resulting in varied bacterial behaviors and characteristics. This study is the first to assess the changes in the biofilm architecture induced by chlorination and the effect of methylparaben (MP) on colony biofilm architecture and conformation using a Mesolens microscope. For this purpose, 2-day-old Stenotrophomonas maltophilia colony biofilms were grown in R2A agar with MP at environmentally relevant (15 μg/L) and in-use concentrations (15000 μg/L), both with and without free chlorine at 5 mg/L. MP exposure induced noticeable changes in the biofilm structure, such as a denser center and the formation of distinct structures like channels within the colonies. The MP-exposed colony biofilms exhibited more pronounced internal structures, evidenced by a higher number of peaks in the intensity profile. Chlorine disinfection significantly reduced the bacterial growth in the colony biofilms, decreasing the colony diameter by more than half. Additionally, the circularity of the colonies was significantly affected when chlorine and MP were simultaneously present. Furthermore, MP compromised drinking water's disinfection and increased S. maltophilia's tolerance to TMP-SMX, resulting in a 26% decrease in the inhibition halo compared to that for the non-exposed counterparts. These findings suggest that MP, even at environmentally relevant concentrations, can significantly impact the architecture of S. maltophilia colony biofilms, affecting their tolerance to chlorine and antibiotics.