Surfactants are extremely important agents to clean and sanitize various environments. Their biocidal activity is a key factor determined by the interactions between amphiphile structure and the target microbial cells. The object of this study was to analyze the interactions between four structural variants of N-alkyltropinium bromide surfactants with the Gram negative Escherichia coli and the Gram positive Listeria innocua bacteria. Microbiological and conductometric methods with a previously described FTIR bioassay were used to assess the metabolomic damage exerted by these compounds. All surfactants tested showed more biocidal activity in L. innocua than in E. coli. N-tetradecyltropinium bromide was the most effective compound against both species, while all the other variants had a reduced efficacy as biocides, mainly against E. coli cells. In general, the most prominent metabolomic response was observed for the constituents of the cell envelope in the fatty acids (W1) and amides (W2) regions and at the wavenumbers referred to peptidoglycan (W2 and W3 regions). This response was particularly strong and negative in L. innocua, when cells were challenged by N-tetradecyltropinium bromide, and by the variant with a smaller head and a 12C tail (N-dodecylquinuclidinium bromide). Tail length was critical for microbial inhibition especially when acting against E. coli, maybe due the complex nature of Gram negative cell envelope. Statistical analysis allowed us to correlate the induced mortality with the metabolomic cell response, highlighting two different modes of action. In general, gaining insights in the interactions between fine structural properties of surfactants and the microbial diversity can allow tailoring these compounds for the various operative conditions.