This paper investigates how the duty ratio influences the production of hydrogen peroxide (H2O2) in an alternating current (AC)-driven plasma jet. H2O2 is a major reactive oxygen species produced by plasma jets in medicine and is a biological important molecule because it can intervene in cellular signaling processes important for disease treatment. In this study, duty percentages were varied from 9% to 72%, which correlated with an increase in the plasma’s dissipated power and UV photon energy density, and consequently an increase in the production of high energy plasma components important for producing H2O2 through electron collision and UV photolysis reactions. Cell media treated with the plasma jet at higher duty ratios correlated with a decrease in the viability of HaCaT keratinocyte skin cells. The AC-driven plasma jet was shown to be optimally operated at a duty ratio of 34% to produce a biocompatible gas temperature of 40°C or below whilst still maintaining relatively high efficiency in H2O2 production. Overall, the data presented in this study might find use for the future of optimisation of the electrical properties of AC-driven plasma jets in plasma medicine.