Hyssopus officinalis L (Hyssop) is a good source of phenolic compounds. However, conventional methods for extraction of these compounds typically take a long time and have relatively low recovery rates. This study focused on cold atmospheric plasma (CAP) pretreatment and investigated its effects on the ultrasound-assisted extraction (UAE) of phenolic compounds from hyssop. Hyssop was treated at dielectric barrier discharge plasma with air and nitrogen gases for 5, 10, and 15 min. Optical emission spectroscopy was used to evaluate present active species in the plasma. The water contact angle changes, cell wall polysaccharides distribution, and structural variations of the treated samples were determined after treatment. Antioxidant activity and total phenolic contents (TPC) of the extracts were also evaluated. The results showed that CAP treatment reduced the contact angle making surface more hydrophilic. Compared with hyssop, overall no significant changes in the basic structure of all treated samples or the formation of new functional groups were recognized. In addition, CAP pretreatment before UAE increased the antioxidant activity of extracts according to the FRAP assay than the un-pretreated sample and conventional solvent extraction method. Also, TPC increased in samples treated with nitrogen plasma.