2-Methyl-3-(ω-mercaptoalkyl)-1,4-naphthoquinones, containing 5, 7, and 9 carbon atoms in the alkyl chain, were synthesized, and the structural and electrochemical parameters of their self-assembled monolayers (SAMs) on gold were studied by in situ Fourier-transform surface-enhanced Raman spectroscopy (FT-SERS) and cyclic voltammetry (CV). We have demonstrated that the alkyl chains of SAM prepared from the concentrated (1mM) substance solution contain a number of gauche defects. Nevertheless, it has been found that the 2-methyl-1,4-naphthoquinone (2-MeNQ) group does not interact directly with gold. In contrast, spectral changes for surface layers prepared from diluted (0.05μM) solutions have indicated flat ring orientation and direct involvement of 2-MeNQ group in the interaction with Au. It has been found that the Au–S stretching band at 291cm −1 considerably increases in intensity and blue-shifts by ∼10cm −1 in the surface spectra of monolayers prepared from low concentration coating solution indicating different Au–S bonding geometry and coordination of sulphur atom with Au. Up-shift in the CO band from 1654 to 1660cm −1 has been detected for the mixed monolayer prepared using shorter alkyl chain diluent, heptanethiol, and indicates absence of stacking interaction between the 2-MeNQ rings as well as involvement of the CO group in the hydrogen bonding with interfacial water. The electrochemical response of 2-MeNQ group buried in the long-chain thiols containing mixed monolayers was found to be controlled by the nature of the diluent terminal group. Thus, replacement of the CH 3 group in dodecanethiol by COOH has induced electrochemical activity of 2-MeNQ group in the mixed SAM.