Biofilms represent the predominant form of microbial life on Earth. They are aggregates of microorganisms embedded in a matrix formed by extracellular polymeric substances (EPS). Detailed information about chemical composition and structure of the EPS matrix is relevant e.g. for the optimization of biocides, of antifouling strategies and for biological wastewater treatment. Raman microscopy (RM) is a capable tool that can provide detailed chemical information about biofilm constituents with spatial resolution of optical microscope. However, the sensitivity of RM is limited. Surface-enhanced Raman scattering (SERS), which enables investigations of biomolecules at very low concentration levels, allows overcoming this drawback. To our knowledge, this paper is the first report on reproducible SERS spectra from different constituents of a multispecies biofilm. We believe that the reproducibility is partly owed to the in situ measurement of the biofilm, while up to now SERS measurements of microbiological samples by RM were carried out after sample drying. We employed colloidal silver nanoparticles for in situ SERS measurements by RM. The achieved enhancement factor of up to 2 orders of magnitude illustrates a high potential of SERS for ultrasensitive chemical analysis of biofilms, including the detection of different components and the determination of their relative abundance in the complex biofilm matrix.