In recent years, plasma biomedicine has attracted increasing attention as a promising method of sterilization and other biomedical applications. This research aims to develop and characterize an argon-based non-thermal atmospheric-pressure plasma jet (APPJ) with an application of sterilization and nail disease treatment. We characterized the electric properties by an electrical measurement system and quantified the active species in APPJ by optical emission spectroscopy (OES). The gas temperature of plasma jet is below 37°C, which is generally free of thermal damage for the human body. The results of OES measurement indicate that this APPJ has a high intensity of hydroxyl radicals (OH∗), which has shown promise in killing microorganisms. The preliminary result from inactivation experiments also show that APPJ efficiently kills bacteria (P. aeruginosa) and yeast (C. albicans). Furthermore, we used bovine hoof as a surrogate model for human nails for penetration tests. The results demonstrated that argon APPJ is good in inhibition of P. aeruginosa (90% in 5 min) and C. albicans (76% in 5 min) on agar plates. In addition, antimicrobial tests showed that approximately 2-log and 4-log reduction can be reached for P. aeruginosa and C. albicans in 5 min, respectively, when the argon APPJ was applied with them deposited at the back of the bovine hoof. We attribute the latter which contradicts with the former may be caused by many possible factors, which include the biofilm formation of the P. aeruginosa on the bovine hoof, the nature and the size difference of these two pathogens.