Herein, subtle changes in the molecular orientations of different thiols upon interaction with soft-landed silver nanoparticles (AgNPs) have been probed using the technique called ambient electrospray deposition Raman spectroscopy (AESD RS). The method collects real-time surface-enhanced Raman scattering (SERS) spectra of analytes as Raman-active AgNPs were electrodeposited on a substrate. We have used p-mercaptobenzoic acid (p-MBA), benzenethiol (BT), and cyclohexanethiol (CHT) as proof-of-concept ligands for understanding variations in molecular orientations as a function of time, immediately after 30–40 s of electrospray of AgNPs. During time-dependent SERS measurements, we observed that the carboxylate (COO–) group of p-MBA preferred a flat orientation on the NP surface upon interaction with the electrosprayed AgNPs, which later transformed into a tilted two-legged standing-up orientation. We also observed a concomitant change in the orientation of the phenyl ring, which transformed from tilted to a flat orientation with respect to the NP surface. We have found that the time of tilting depends on the concentration of the analyte used for analysis. Additional information on the orientation flipping of thiols was achieved by performing real-time SERS experiments on other thiol derivatives such as CHT and BT. In the case of BT, SERS intensity variations were similar to p-MBA, which were attributed to the changes in orientation. However, in the case of CHT, such SERS intensity variations were absent. Time-dependent SERS spectra of p-MBA, BT, and CHT suggested that the interaction between the π-cloud of the phenyl ring and AgNPs could be one of the triggering factors for such orientation flipping of thiols on NP surfaces.