The variation in the biological function of proteins plays an important role in plasma medicine and sterilization. Several non-thermal plasma sources with different feeding gases are used worldwide for plasma treatment, including dielectric barrier discharge (DBD) and atmospheric-pressure plasma jet (APPJ) as the most commonly used sources. Therefore, in the present work, we used both DBD and APPJ plasma sources with N2 and air as feeding gases to evaluate the effects on the structural, thermodynamic, and activity changes of enzymes. In the current work, we used lysozyme as a model enzyme and verified the structural changes using circular dichroism (CD), fluorescence, and X-ray crystallography. In addition, we investigated the lysozyme thermodynamics using CD thermal analysis and changes in the B-factor from X-ray crystallography. The results showed that lysozyme activity decreased after the plasma treatment. From these analyses, we concluded that N2-feeding gas plasma disturbs the structure and activity of lysozyme more than Air feeding gas plasma in our experimental studies. This study provides novel fundamental information on the changes to enzymes upon plasma treatment, which has been absent from the literature until now.