Despite being renowned for its volatiles, the data on the toxicity of the essential oil of lemon balm (Melissa officinalis L., Lamiaceae) is rather limited compared to its solvent/water-soluble extractibles. In this study, the aerial parts essential oil of M. officinalis, with over 130 constituents identified herein, 26 of which detected for the first time, was investigated for acute oral toxicity in BALB/c mice. The oil, composed of predominantly monoterpene aldehydes, citronellal (21.2–21.8%), neral (17.8–18.4%), and geranial (22.9–23.5%), which were assayed in parallel with the oil in some tests, induced significant changes in animal behavior, as well as altered biochemical parameters reflecting liver and kidney functions. Different pathological changes in the stomach, duodenum, liver, and kidneys were detected when the oil was administered in doses higher than 1 g kg−1. A depletion in the liver/kidney antioxidant capacities and an increased rate of lipid peroxidation was noted for animals treated with lemon balm oil. The calculated value of the oral LD50 in BALB/c mice (2.57 g kg−1) infers that the essential oil is only moderately toxic. Display omitted •Twenty-six compounds are reported here for the first time as constituents of M. officinalis essential oil.•Oral application of lemon balm essential oil causes significant changes in mice behavior, and liver and kidney functions.•Above 1 g kg−1 (p.o.), different pathological changes in the stomach, duodenum, liver, and kidneys were detected.•The essential oil produced a depletion of liver/kidney antioxidant capacities and increased the rate of lipid peroxidation.•The determined oral LD50 in BALB/c mice (2.57 g kg−1) suggests that the essential oil is only moderately toxic.