Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by L-ascorbate. L-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM L-ascorbate. However, brief pre-treatment with a pharmacological dose (250–750 μM) of L-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of L-ascorbate was completely abolished by a prolonged 4 h pre-incubation with L-ascorbate (500 μM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2′,7′-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and L-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.