A femtosecond laser can be used to fabricate microstructures on a silicon microcantilever surface with high precession and minimal sidewall defects. The aim of this study is to investigate the effect of the creation of microgrooves and sub-microgrooves on the resonance frequency, quality factor, and spring constant of a silicon microcantilever. A single pass of a femtosecond laser with a wavelength of 1026 nm was used to fabricate microgrooves on the microcantilever surface. Different numbers of microgrooves were fabricated on each microcantilever using the femtosecond laser micromachining technique. The separation distance between the center of the two microgrooves was 7 μm. The microstructure of the fabricated microgrooves was investigated through field emission electron microscopy. The resonance frequency increased with the number of microgrooves, but the quality factor of the patterned microcantilever was higher than that of the unpatterned microcantilever. The spring constant increased with the number of microgrooves, increasing from 18.96 to 38.04 mN/m for microcantilevers with 1 and 7 microgrooves, respectively.