The power of photothermal spectroscopic imaging to visualize antimicrobial interaction on the surface of individual bacteria cells has been demonstrated on the model system Bacillus subtilis and vancomycin using mid-infrared photo-induced force microscopy (PiF-IR, also mid-IR PiFM). High-resolution PiF contrasts obtained by merging subsequent PiF-IR scans at two different illumination frequencies revealed chemical details of cell wall destruction after 30 and 60 min incubation with vancomycin with a spatial resolution of ≈ 5 nm. This approach compensates local intensity variations induced by near-field coupling of the illuminating electric field with nanostructured surfaces, which appear in single-frequency contrasts in photothermal imaging methods, as shown by [Anindo et al., J. Phys. Chem C, 2025, 129, 4517]. Known spectral shifts associated with hydrogen bond formation between vancomycin and the N – acyl – D – Ala4 – D – Ala5 termini in the peptidoglycan cell wall have been observed in chemometrics of PiF-IR spectra from treated and untreated Bacillus subtilis harvested after 30 min from the same experiment. Spectral signatures of the vancomyin interaction have been located in the piecrust of a progressing septum with ≈ 10 nm resolution using PiF contrasts of three selected bands of a PiF-IR hyperspectral scan of an individual Bacillus subtilis cell harvested after 30 min incubation. Our results are complemented by a discussion of imaging artifacts and the influence of parameter settings supporting further development towards standardization in the application of PiF-IR for visualizing the chemical interaction of antibiotics on the surface of microbes with few nanometer resolution.