We have investigated the interaction of surfactin with a monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air-water interface as a function of time, following its injection into the sub-phase, using non-linear Sum Frequency Generation (SFG) vibrational spectroscopy and Infrared Reflection Absorption Spectroscopy (IRRAS). SFG resonances from the phospholipid and from the surfactin were distinguished from each other by using selective deuteration. The surface pressure at the interface was measured concurrently for up to 8 h. After an induction period, the spectra from the lipid diminished and those of surfactin gradually appeared whilst at the same time the surface pressure increased. However, eventually the surfactin signals disappeared and those of the lipid reappeared. Although the SFG spectra of the lipid disappeared at intermediate times, the IRRAS spectra of the lipid were always present at the interface. Variation in the temporal SFG behaviour was investigated as the pH of the sub-phase, the initial surface pressure of the lipid, and the surfactin concentration were changed. Samples of the surface film were transferred onto mica substrates at selected times along the temporal profile and imaged by Atomic Force Microscopy - nano Infrared Spectroscopy (nano-IR). A model is proposed to account for the results from the four different experimental techniques used.