Among the anthropogenic sources of pollution, accumulation of plastic polymers in aquatic ecosystems is scaling at unprecedented rates and emerging as a new niche for bacterial colonization and horizontal gene transfer (HGT). The current study focuses on determining the ability of bacteria to acquire plasmid DNA from the extracellular environment under exposure to different treatments (soil, CaCl₂ salt solution, soil plus CaCl₂, Escherichia coli cell-free extract, and plastic debris) that simulate possible conditions experienced by microorganisms in natural environments. The transformation frequency of two plasmids (pACYC:Hyg and pBAV-1k) was tested following two experimental approaches: single species microcosm of E. coli cells (SSM) and bacterial consortium microcosm (BCM) of strains isolated from freshwater ecosystems. Plastic fragments (with consistent results obtained using polypropylene) proved to be remarkably efficient in increasing the bacterial competence towards plasmid DNA uptake as compared to the other conditions. Moreover, the effects of different plastic polymers and four incubation conditions on bacterial DNA transformation were analyzed to gain deeper insight into the exchange of genetic material. Our findings from both experimental approaches demonstrate that simultaneous incubation of microorganisms, plasmids, and plastic fragments enhances the bacterial ability to uptake plasmids and to express genes required for survival under stress conditions. The two microcosm models prove to be promising tools to mimic natural transformation events leading to the dissemination of antibiotic-resistant genes via HGT in the environment.