Mid-infrared spectroscopy in conjunction with Functionally-Enhanced Derivative Spectroscopy (IR+FEDS) is a powerful analytical tool for the improvement of analysis of microorganism IR spectra. The objective of this research is to characterize the outer surface of two Helicobacter pylori strains by IR+FEDS. This work is a key stage for the study of cell-cell and cell-surface interactions between microorganisms, as well as, for polymicrobial biofilm characterizations where H. pylori species are involved. Artificial bacterial biofilms were deposited on ultrafiltration cellulose membranes covalently modified by insertion of one spectral marker and used as sensing surface for analysis of bacterial biolayers. Biolayers were analyzed using an infrared spectrophotometer with ATR. Data were analyzed by classic procedures and by deconvolution based on FEDS transform. It is concluded that, for correct application of technique is required a minimum amount of noise in the working spectra which can be achieved by simple smoothing algorithms; in addition, reproducibility must be warranted by the implementation of standardized protocols and the use of an appropriate number of samples. It is concluded that in addition to typical signals associated with the IR spectrum of microorganisms, by FEDS, a better and more detailed description of the outer membrane of H. pylori biofilms can be performed. In particular, it is concluded that the detecting and monitoring of cysteine-rich proteins can be satisfactorily performed by IR+FEDS.