The substituent effect on structure and surface activity of mono- and disubstituted N-methylpyridinium salts was investigated by means of Raman, infrared and surface-enhanced Raman spectroscopy (SERS). The significant differences observed in Raman and infrared spectra have been correlated with marker bands assigned to in-plane and out-of-plane vibrations, respectively. This vibrational analysis, complemented by quantum chemical calculations (B3LYP/6-311++G(d,p)) was a basis for investigation of the surface activity of the studied compounds. Significant differences in their SERS spectra related to the enhancement mechanism and adsorbate orientation have been observed and analyzed. Copyright © 2012 John Wiley & Sons, Ltd.