Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on cell viability and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for >2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, >20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-5-fold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.