The activity of the dermaseptine S4 derivative S4(1-16)M4K against 6h-old Pseudomonas fluorescens biofilms has been assessed by using the Attenuated Total Reflectance - Fourier Transform InfraRed (ATR-FTIR) spectroscopy. The ATR-FTIR fingerprints of bacteria were monitored in situ and in real time during 24-hour treatment with the antimicrobial peptide (AMP) by using an ATR-FTIR flow cell. After antimicrobial peptide treatment biofilms were analyzed by epifluorescence microscopy using the live/dead bacterial viability kit in order to determine bacterial surface coverage and the viability of biofilm cells. After exposure to the peptide, major and rapid changes or in contrast no change over time were observed in ATR-FTIR fingerprints of biomolecules revealing major biochemical and physiological changes, adsorption peptides on the bacterial membranes, bacterial detachment, bacterial regrowth or in contrast inhibition of biofilm growth. This study shows that the ATR-FTIR technique can be a valuable tool for in situ monitoring the activity of AMPs against sessile bacteria.