Xenobiotics are a range of chemicals and compounds that are not present in biological systems, i.e., antibiotics and drugs, aflatoxins, heavy metals, pesticides, microplastics (MPs), and others. MPs are widespread contaminants that are highly persistent in the environment and present in matrices to which humans are extensively exposed, including food and beverages. MP ingestion occurs in adults and children and is becoming an emerging public health issue. The gastrointestinal system is the most exposed to MP contamination, which can alter its physiology starting from changes in the microbiota. The toxicity of these compounds is mediated by the gut microbiota, which have a variable response depending on differences in microbiota population and activity. In these terms, there is an impellent need to elaborate and standardize reliable risk assessment procedures to test xenobiotics directly on human gut microbiota. This study investigates, by an omics approach, the impact of a single intake of a mixture of polyethylene (PE) and polystyrene (PS) MPs on the ecology and metabolic activity of the colon microbiota of healthy volunteers in an in vitro intestinal model. PE and PS microplastics were pooled together in a homogeneous mix, digested with the INFOGEST system, and fermented with MICODE (multi-unit in vitro colon model) at a maximum load of 0.166 g, that by the literature corresponds to the intake of food-derived microplastics of a single meal. The results demonstrate that the MP mix induced opportunistic bacteria overgrowth (Enterobacteriaceae, Desulfovibrio spp., Clostridium group I, and Atopobium --Collinsella group) and a contextual reduction in the abundances of all the beneficial taxa analysed, with the sole exception of Lactobacillales. This microbiota shift was consistent with recorded changes in bacterial metabolic activity, expressed as the higher production of indoles and phenols and lower production of short chain fatty acids.