Multispectral digital lensless holographic microscopy (MDLHM) operating with second-harmonic illumination is shown. Added to the improvement of the spatial resolution of the previously reported MDLHM operating with near-infrared illumination, this second-harmonic MDLHM shows promise as a tool to study the behavior of biological samples under a broad spectral illumination. This illumination is generated by focusing a highly spatially coherent ultrashort pulsed radiation into an uncoated Type 1 β-BaB2O4 (BBO) nonlinear crystal. The second-harmonic MDLHM allows achieving multispectral images of biological samples with enhanced micrometer spatial resolution. The illumination wavelength of the second-harmonic MDLHM can be tuned by displacing a focusing optics with respect to a pinhole; spatially resolved information at different wavelengths of the sample can then be retrieved.