New surface active ionic liquids (SAILs), based on cholinium cations and dodecylbenzenesulfonate as anion, have been synthesized and their potential application as antimicrobial colonization agents on cultural heritage (CH)stone materials investigated. The biocidal activity and antifouling capabilities were, preliminarily, evaluated by a screening on pure Gram (+) and Gram (−) bacteria strain cultures, yeasts, hyphomycetes and single-celled algae. Tests on stone materials (marble and tufa) vs. a stabilized community, constituted by a mixture of microbial strains, revealed that some SAILs display both antimicrobial and preventive antibiofilm action against new colonization. Analogous tests have been performed on the cholinium@halide precursors.