The use of medicinal plants in the United States is increasing. Holy basil (Ocimum tenuiflorum L. and Ocimum gratissimum L.), a medicinal herb native to India, has become increasingly popular for its therapeutic benefits. Traditionally, holy basil has been used to promote longevity by reducing stress and restoring balance to the body. Because it is easy to grow and adapts to a wide range of growing conditions, there is great potential for holy basil production in the southeastern United States. The purpose of this study was to evaluate holy basil varieties for harvestable weight and essential oil content. Fourteen varieties of holy basil were grown during the 2015 and 2016 seasons and compared. The main active compound in the holy basil essential oil fraction, eugenol, was quantitated and compared for each variety, because it is believed to be responsible for many of the health-promoting effects. Overall, there were significant differences in harvestable weights and essential oil yields among the varieties, and a significant effect of growing season. The eugenol content was highly variable among the varieties examined, with higher eugenol contents in 2016 than in 2015. The variety that had the overall highest yield, essential oil content, and eugenol concentration was PI 288779, a USDA accession, suggesting its use in future breeding research.