An inactivation mechanism of A549 cancer cells is studied by using a dielectric barrier discharge (DBD) plasma needle. The influence of oxygen concentration, which is injected into helium plasma afterglow region through a stainless steel tube, is investigated. The neutral red uptake assay provides a qualitative observation of morphological differences between the dead cells and the viable cells after plasma treatment and a quantitative estimation of cell viability under different conditions. In the treatment process at a fixed power of 24 W, the inactivation efficiency of helium-oxygen plasma depends mainly on the exposure time and percentage of added oxygen in helium plasma. Experimental results show that the best parameters of the process are 150 s treatment time, 800 mL/min He with 3% O2 addition and separation of needle-to-sample 3 mm. According to the helium-oxygen emission spectra of the plasma jet, it is concluded that the reactive species (for example, OH and O) in the helium-oxygen plasma play a major role in the cell deactivation.