In the present study, deep eutectic solvent (DES) was prepared by combining choline chloride and glycerol and is used as a green alternative extraction media of detrimental organic solvents. The DES and aqueous methanol were used for the ultrasound-assisted extraction (UAE) of bioactive compounds from Mentha arvensis, which have remarkable medicinal values. The optimization of extraction parameters during phytochemical evaluation was done by response surface methodology. The extraction yield obtained with DES was significantly higher than that achieved using aqueous methanol. The total phenolic contents (TPCs) 117 mg GAE/g, total flavonoids contents (TFCs), 80 mg QE/g and 2, 2-diphenyl-1- picrylhydrazyl (DPPH) radical inhibition 94% was obtained using DES. The antimicrobial activities of extracts were evaluated against S. aureus, E. coli (bacterial strains), F. solani and A. niger (fungal strains) by well diffusion method. The extracts were characterized using LC-MS and showed that chrysin, p-coumaric acid, naringenin, scopoletin, phenylpyruvic acid, pinocembrin, hesperidin, carnosic acid and caffeic acid were the main bioactive components of Mentha arvensis. The DES has shown considerable high extraction yield, which ultimately relates to the higher TPCs, TFCs and DPPH assay compared to aqueous methanol. The DES with extensive hydrogen bonding is of vital importance in the perspective of the environment than traditionally used solvents.