A portable atmospheric pressure resistive barrier plasma (RBP) device is designed, constructed, and characterized for plasma surface treatment procedures applied in biomedical applications. The design and construction aspects of the RBP plasma device are presented including the electrode configuration, electrical, cooling, and gas flow rates. The RBP device can operate in both dc (battery) as well as in standard 60/50 Hz low frequency ac power input. The RBP device can function effectively in both direct and indirect plasma exposure configurations depending on the type of treatment targets. The portable RBP device is characterized for plasma jet exit velocity, plasma temperatures, and reactive nitrogen species (RNS) using laser shadowgraphy, emission spectroscopy, and gas analyzer diagnostics. We have measured the average velocity of the plasma jet to be 150–200 m/s at 1 cm from the probe end. The gas temperature which is equivalent to the rotational (Trot) temperatures of the plasma is measured by simulation fitting the experimental emission spectra. A high-temperature ceramic fiber-insulated-wire thermocouple probe is used to measure the temperatures of the downstream jet after 2 cm where the plasma emission drops. Addition of external cooling unit brought the temperatures of reactive species and other gases close to room temperature. The spatial concentrations of the reactive oxygen species from the plasma jet tip are measured at 5 cm distance from the electrode. The nitric oxide level is measured to be in the range of 500–660 ppm and it drops to ∼100 ppm at 60 cm. The ppm values of nitric oxides after the cooling unit are observed to be at the same order of magnitude as the plasma jet. The preliminary results on the effectiveness of the portable RBP device for bacterial inactivation as well as the effects of indirect exposure of the portable RBP device on monocytic leukemia cancer cells (THP-1) are briefly presented.