Mismanagement of plastic waste has contributed to plastic pollution in marine and terrestrial ecosystems. Therefore, farm animals are likely to consume microplastic (MP) contaminated feed. However, the interactions of MP with the ruminal microbial ecosystem remain poorly understood. This study investigated the interaction of MP within the ruminal ecosystem in vitro using the Hohenheim Gas Test. Different MP variants were applied, reflecting different combinations of five MP species (polylactide, polyhydroxy butyric acid, high-density polyethylene, polyvinyl chloride, polypropylene), in two particle size ranges (<125 μm; 125-500 μm) and increasing dosages (from 0 to 70 mg /incubation cylinder) together with ruminal fluid and hay or barley as substrates. Cumulative gas production, pH and dry matter disappearance were determined before analyzing volatile fatty acids, metaproteomics and metabolomics. In the presence of MP, cumulative gas production decreased regardless of the MP 63 species, dose or particle size, while total dry matter degradation increased. Microbial proteins in barley incubations showed lower Bacteroidetes and increasing Firmicutes abundance in the presence of MP and increased activities of the protein groups `replication and repair`, and `translation`, but decreased activities of `carbohydrate metabolism and transport` and `amino acids metabolism`. The data indicated that MP, regardless of their species and particle size, interact with the ruminal microbiome and may be partially degraded in vitro. This suggests a reduction of MP size in the rumen thereby increasing the likelihood to penetrate animal tissues. Future research must confirm the findings in vivo and determine their consequences for animal health and consumer safety.