This study was carried out to investigate the phytochemical screening, antioxidant activity, and anxiolytic-like effects of the ethanolic extract of Melissa officinalis L. (MO) (Family of Lamiaceae). The antioxidant activity of MO was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. The antioxidant activity and phytochemical screening were performed on the ethanol extract of MO and then was orally administered to rats at doses of (30, 300, and 500 mg/kg daily) during the experimental period of 10 days for behavioral tests by using the elevated plus maze (EPM), and biochemical studies. Phytochemical screening revealed the presence of alkaloids, flavonoids, saponins, steroids, triterpenoid and tannins. The phenol concentration was (46.90 mg EAG/g DM), and flavonoids were found to be (15.06 mg EQ/g DM). DPPH radical scavenging assay showed potent antioxidant power with IC50 values of 15.44 µg/ml and ABTS (137.45 µg/ml). The sub-chronic administration of Melissa officinalis L. ethanol extract increased significantly the percentage of entries and the time spent on the open arms compared with a control group. The levels of malondialdehyde (MDA) and nitric oxide (NO) were significantly reduced by MO ethanol extract. The present study reveals that Melissa officinalis L. can be used as a potential source of natural antioxidants to prevent oxidative stress, and suggests that it possesses anxiolytic-like effect.