Microplastic (MP) pollution is a growing concern in every known ecosystem. However, MP presence in groundwaters and the ecological impact they can have on groundwater fauna is still poorly investigated. Here, we assess the presence of MPs in three Italian groundwater bodies, comprising two karst caves and two monitoring bores of a saturated alluvial aquifer. In addition to water samples, groundwater invertebrates were collected to assess their potential ingestion of MPs. For water samples, chemical characterization of polymers was done by Fourier Transform Infrared Spectroscopy (FTIR) 2D imaging, while fauna samples were analyzed with a tandem microscopy approach (fluorescence microscopy and FTIR). The abundance of MPs in water samples varied from 18 to 911 items/L. The majority of MPs were fibers (91%), with a mean size in the range of 100 µm and 1 mm. Black, red, and blue were the most abundant colors (30%, 25%, and 19%, respectively). The most abundant polymer was artificial/textile cellulose (65%), followed by PET (21%). MPs were found in every groundwater taxon. Pellets were the most abundant in each specimen (87% on average), while the largest were fragments, with a mean dimension of 26 µm. Cellulose was found to be the most abundant polymer (51%). This study is the first to highlight the presence of MPs ingested by groundwater fauna. Further investigations are urgently required to assess the potential ecological impact MPs can have on the resident fauna in these sensitive ecosystems.