The cultivation of lemon balm (Melissa officinalis L.) is necessary for pharmaceutical and cosmetic production. The aim of our work was to investigate the effect of the plant density as well as the harvesting method on the yield and quality characteristics of M. officinalis, which is used as a source of polyphenols and essential oil in the pharmaceutical, food, and cosmetic industries. The field experiment was carried out in two growing seasons (2019 and 2020). The experiment was set up as a 2-factor experiment; the factors studied were the plant spacing (30 × 30 cm or 40 × 40 cm) and the harvesting method (single harvesting or double harvesting). The lemon balm herb was cut twice (in mid-July and early September) or once (in early September) depending on the combination. The air-dried leaves were subjected to laboratory tests to determine the essential oil, total tannin, and total flavonoid contents. The yield of lemon balm in the individual years in the study depended more on the harvesting method than on the plant density. The essential oil content was 0.25–0.38% depending on the plant density. Considering the yields of the fresh and air-dried herb and leaves, essential oil yield and oil, and tannin and flavonoid contents, a higher plant density (40 × 40 cm) is a better agronomic option than a lower density (30 × 30 cm). Harvesting the herb twice proved to be more efficient in terms of quantity and quality than a single harvest.