L.) species are used as medicinal plants due to their essential oils exhibiting specific biological activity. The present work demonstrated that both the variety and season/conditions of cultivation had a significant effect on (i) the produced amount (extraction yield), (ii) qualitative, as well as (iii) quantitative profile of basil essential oil. Among studied basil varieties, a new variety, ‘Mánes’, was characterized for the first time. Based on our quantitative evaluation of GC-MS profiles, the following chemotypes and average concentrations of a main component were detected in the studied basil varieties: ‘Ohře’, ‘Lettuce Leaf’, ‘Purple Opaal’, ‘Dark Green’ (linalool, 5.99, 2.49, 2.34, 2.01 mg/mL, respectively), and ‘Mammolo Genovese’, ‘Mánes’, ‘Red Rubin’ (eucalyptol, 1.34, 0.96, 0.76 mg/mL, respectively). At the same time, when considering other compounds identified in GC-MS profiles, all the studied varieties, except from ‘Lettuce Leaf’, were methyl eugenol-rich with a strong dependence of the eugenol:methyl eugenol ratio on the seasonal changes (mainly solar irradiation, but also temperature and relative humidity). More complex and/or variable (depending on the season and cultivation) chemotypes were observed with ‘Lettuce Leaf’ (plus estragole, 2.27 mg/mL), ‘Dark Green’ (plus eucalyptol, 1.36 mg/mL), ‘Mammolo Genovese’ (plus eugenol, 1.19 mg/mL), ‘Red Rubin’ (plus linalool and eugenol, 0.46 and 0.56 mg/mL, respectively), and ‘Mánes’ (plus linalool and eugenol, 0.58 and 0.40 mg/mL, respectively). When considering superior extraction yield (ca. 17 mL·kg−1
, i.e., two to five times higher than other examined varieties) and consistent amounts (yields) of essential oil when comparing inter-seasonal or inter-year data (RSD and inter-year difference in mean yield values ˂2.5%), this new basil variety is very promising for use in the pharmaceutical, food, and cosmetic industries.