In this letter we report a fast and easy method which could be used for initial screening of multispecies-biofilm development on putative new dental implant materials. Most staining methods require numerous washing steps that can result in detachment of loosely bound biofilms and therefore falsify the results. Thus, we used glutaraldehyde fixation, which induces autofluorescence through bacterial membrane protein cross-linking and concurrently stabilizes the biofilm structure. We analyzed the biofilms with nonlinear laser scanning microscopy and were able to (I) evaluate the multispecies-biofilm growth and (II) distinguish between bacterial species based on different two-photon autofluorescence intensities. © 2016 by De Gruyter.