Aims This research aims to develop strongly adherent and mature model biofilms (on a 20 cm(2) polystyrene surface) for two pathogenic species, i.e. Listeria monocytogenes and Salmonella Typhimurium. These model biofilms can be used as standards to study biofilms or to study/compare the influence of different inactivation technologies. Methods and Results Conclusions Three influencing factors on the formation of biofilms are investigated, i.e. growth medium, incubation temperature and incubation time, which are three easily controllable environmental factors. Optical density measurement and plate counts were used to evaluate the adherence and the maturity of the biofilms, respectively. Confocal laser scanning microscopy was used to verify most important findings obtained with previously mentioned assays. Results indicated that mature and strongly adherent L. monocytogenes biofilms are obtained following 13 h of incubation at 30 degrees C with BHI as growth medium. For S. Typhimurium, an incubation period of 19 h at 25 degrees C was required with 20-fold diluted TSB as growth medium. Based on previously mentioned assays, a protocol for the formation of reproducible model biofilms was obtained. Significance and Impact of the Study The developed model biofilms can be applied as a standard to study biofilms (in different research fields) and their subsequent inactivation by different methods. In addition, the results of this study could be used to control biofilm formation (e.g. by setting a maximum allowed surface temperature).