Antimicrobial peptides (AMPs) free in solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied and various models ranging from Pore formation to Carpet-like mechanism were proposed. Surface immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, SFG-microscope, which is composed of a sum frequency generation (SFG) vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide-bacteria interaction. It was found that upon bacteria contact, the surface immobilized peptides became bent and killed bacteria. This research demonstrated that the SFG-microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure-activity relationships of surface immobilized AMPs.