In vitro treatment of hepatic malignant cells with plasma‐activated medium conjugated with gemcitabine was investigated with a He plasma jet generated by a two‐ring dielectric barrier discharge (DBD) reactor. The DBD reactor was operated by high‐voltage pulses of alternating polarity (frequency: 20 kHz; pulse width: 4 µs). The electron density of plasma plume was estimated to be 9×1012 to 10×1012 cm−3, and the jet temperature was around 36℃. The plasma jet exhibited the inhibitory effects on the growth of hepatic malignant cells via the generation of reactive oxygen species, which could be potentially applicative in anticancer therapies. The optimization of the operating parameters and configuration of the plasma jet would help increase the possibility in practical cancer treatments.