Sample ablation by pulsed lasers is one option for removing material from a sample surface for in situ depth profiling during imaging mass spectrometry, but ablation is often limited by laser-induced damage of the remaining material. A preliminary evaluation was performed of sub-100-fs, 800 nm pulsed laser ablation for depth profiling of bacterial biofilms grown on glass by the drip flow method. Electron and optical microscopy were combined with laser desorption vacuum ultraviolet postionization mass spectrometry to analyze biofilms before and after ablation. Ultrashort laser pulses can ablate 10–100μm thick sections of bacterial biofilms, leaving behind a layer of lysed cells. However, mass spectra from intact and ablated biofilms doped with antibiotic are almost identical, indicating little chemical degradation by ablation. These results are consistent with prior observations from laser surgery and support the use of ultrashort pulse laser ablation for minimally disruptive depth profiling of bacterial biofilms and intact biological samples.