The aim of this study was to clarify the heights and spatial relationships of the facial muscles acting on the nasolabial fold (NLF) by dissection and three-dimensional microcomputed tomography for use in aesthetic treatments. This study used 56 specimens from 34 embalmed adult Korean. A reference line (RF) was set to imitate the NLF after removing the skin, from the superior point of the alar facial crease to the lateral point of the orbicularis oris muscle at the level of the corner of the mouth. The heights and spatial relationships of the facial muscles along the RF could be categorized into five main patterns. The dominant pattern was that the levator labii superioris alaeque nasi muscle (LLSAN), levator labii superioris muscle (LLS), zygomaticus minor muscle (Zmi), and zygomaticus major muscle (Zmj) were on the medial third, medial half, middle third, and lateral third of the RF, respectively. In micro-CT imaging, beneath the skin of the medial half of the NLF, the LLSAN and Zmi fibers inserted into the dermis of the NLF and adjacent to the NLF. Beneath the skin of the middle third of the NLF, the Zmi fibers were found before the muscle inserted into the dermis of the NLF and adjacent to the NLF. Beneath the skin of the lateral third of the NLF, the lateral margin of the orbicularis oris muscle and some Zmj fibers were found at the location of the NLF. The present study utilized dissections and micro-CT to reveal the general pattern and variations of heights and spatial relationships of the facial muscles passing beneath the NLF. These findings will be useful for understanding which muscles affect specific parts of NLFs with various contours, for reducing the NLF in aesthetic treatments, and for reconstructing the NLF in cases of facial paralysis.