Benthic diatoms and bacteria often co-operatively build up phototrophic epilithic biofilms. Studying the properties and contributions of the individual partners requires the establishment and maintenance of axenic cultures of the involved organisms. Axenification of biofilm organisms is often challenging, because bacteria as well as diatom cells are embedded in a matrix of extracellular polymeric substances (EPS). Due to this mucilage, the cells stick together and also are less affected by antimicrobial substances. Here we describe a short and feasible protocol for culture axenification, which was successfully applied to cultures of the benthic diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea. Our protocol includes treatment of the cultures with the antibiotic imipenem and might also be useful for the purification of other cultivated diatom strains. Once axenified, diatom cultures often decay after a certain life span. Our protocol is especially useful to re-establish axenic cultures from co-cultures of diatoms with their accompanying bacteria (also referred to as xenic cultures).