Since numerous characteristic absorption lines caused by molecular vibration exist in the mid-infrared (MIR) wavelength region, selective excitation or selective dissociation of molecules is possible by tuning the laser wavelength to the characteristic absorption lines of target molecules. By applying this feature to the medical fields, less-invasive treatment and non-destructive diagnosis with absorption spectroscopy are possible using tunable MIR lasers. A high-energy nanosecond pulsed MIR tunable laser was obtained with difference-frequency generation (DFG) between a Nd:YAG and a tunable Cr:forsterite lasers. The MIR-DFG laser was tunable in a wavelength range of 5.5–10 μm and generated laser pulses with energy of up to 1.4 mJ, a pulse width of 5 ns, and a pulse repetition rate of 10 Hz. Selective removal of atherosclerotic lesion was successfully demonstrated with the MIR-DFG laser tuned at a wavelength of 5.75 μm, which corresponds to the characteristic absorption of the ester bond in cholesterol esters in the atherosclerotic lesions. We have developed a non-destructive diagnostic probe with an attenuated total reflection (ATR) prism and two hollow optical fibers. An absorption spectrum of cholesterol was measured with the ATR probe by scanning the wavelength of the MIR-DFG laser, and the spectrum was in good agreement with that measured with a commercial Fourier transform infrared spectrometer.