Nanoparticles were proposed as antibacterial cement admixtures for the production of cement-based composites. Nevertheless, the standards for evaluation of such admixtures still do not indicate which model organisms to use, particularly in regard to the further application of material. Apart from the known toxicity of nanomaterials, in the case of cement-based composites there are limitations associated with the mixing and dispersion of nanomaterials. Therefore, four nanooxides (Al2O3, CuO, Fe3O4, and ZnO) and seven microorganisms were tested to initially evaluate the applicability of nanooxides in relation to their further use in cement-based composites. Studies of nanoparticles included chemical analysis, microbial growth kinetics, 4- and 24 h toxicity, and biofilm formation assay. Nanooxides showed toxicity against microorganisms in the used concentration, although the populations were able to re-grow. Furthermore, the effect of action was variable even between strains from the same genus. The effect of nanoparticles on biofilms depended on the used strain. Gathered results show several problems that can occur while studying nanoparticles for specific further application. Proper protocols for nanomaterial dispersion prior the preparation of cement-based composites, as well as a standardized approach for their testing, are the fundamental issues that have to be resolved to produce efficient composites.