Electrochemical SERS (E-SERS) was used for the first time to study the interfacial behavior of a class of pyridinium-based biodegradable ionic liquids at a silver nanoparticle (AgNP) electrode surface. An isomeric series of ionic liquids (IL) based on 3-butoxycarbonyl-1-methylpyridinium bis(trifluoromethanesulfonyl)imide were prepared, which have demonstrable biodegradability. It was found that all four of the isomeric ionic liquids studied exhibited excellent electrochemical stability as binary mixtures combined with methanol, with the absence of any specific redox processes occurring over nearly 3.0 V of applied potential. Normal Raman measurements of the neat isobutyl IL showed a signal rich in vibrational features, with strong contributions from both the anion and the bulky organic cation. E-SERS of the neat isobutyl IL was shown to exhibit excellent potential stability, with no potential-induced orientational change at the metal surface. When the ionic liquids were prepared as methanolic binary mixtures, dissociation of the IL ions was observed, and only the organic cation was shown to adsorb at the Ag/solution interface. The nature of the substituent on the ester group of the IL series was observed to have a significant effect on the orientation of the cation on the metal surface, based on the application of the metal surface selection rules combined with computational data. Notably, the isobutyl and sec-butyl isomers were observed to have an orientation wherein the pyridinium ring was oriented perpendicular to the surface, while the tert-butyl and n-butyl isomers were observed to have an orientation wherein the pyridinium ring was lying flat on the metal surface.