Surface-enhanced Raman scattering (SERS) is a powerful technique for the analysis of a variety of molecules and molecular structures. Due to its great complexity, the acquisition of detailed molecular information from biological organizations such as bacteria is still a challenging task. SERS can provide valuable information once silver or gold surfaces can be brought in close contact with the biological organization. Because several experimental parameters can affect SERS spectra of bacteria, the experimental conditions must be well defined for comparable and reproducible results. The influence of experimental parameters, such as the type of noble metal, size, and aggregation properties of nanoparticles, and the wavelength of the laser light on the SERS of E. coli and B. megaterium are examined. It is demonstrated that the impact of these parameters could be enormous and a standard protocol must be developed depending on the goal of the study.