The protein inventory of the anaerobic pathogen Clostridioides difficile grown in colony or aggregate biofilms was investigated in a comprehensive LC-MS/MS approach. C. difficile has a versatile metabolism and is perfectly equipped to survive and persist inside the mammalian intestine. However, knowledge on how C. difficile exactly resides inside the intestine is still scare. Aggregate or biofilms attached to the epithelium are discussed as possible growth forms and have become a focus in research. However, recent data suggest that the choice of the model strain, timepoint of analysis and the biofilm model significantly impact the outcome of biofilm experiments leading to contradictory results. To address some of these issues, a comprehensive proteomics approach was applied to investigate the metabolism of C. difficile strain 630erm grown in colony or aggregate biofilms. Besides verification of recent findings, a comprehensive overview on the differential expression profile of C. difficile’s cell surface proteins as well as its energy and stress metabolism during the two different forms of biofilm growth could be provided. Additionally, regulatory circuits that were activated in the two different biofilm models were analysed whereby RpoN was determined to be an important regulator of aggregate biofilm formation in C. difficile.