Background Nonthermal atmospheric pressure sterilization is one of the suggested and efficient techniques to hinder the spread of illnesses. Reactive species such as oxygen, hydroxyl, and other radicals play a prime role in the mechanism of plasma sterilization. Staphylococcus epidermidis is the most prevalent cause of primary bacteremia and infections of indwelling medical instruments. The ability to induce disease is related to its natural niche on the skin and capability to adhere and form a biofilm on foreign surfaces. Klebsiella pneumoniae is a zoonotic pathogen frequently isolated from infections related to the presence of bacterial biofilm on devices, such as catheters, which are responsible for loss of patients’ health. Objective S. epidermidis and K. pneumoniae are being combated due to their high frequency of occurrence and ability to form biofilms as survival and virulence characteristics. These particular benefits impose a significant financial burden on hospitals. Materials and methods In this study, the nonthermal plasma treatment induced by surface dielectric-barrier discharge was used to destruct the developed biofilm formed by clinical S. epidermidis and K. pneumoniae isolated from clinical cases of small ruminants. The biofilms were induced in vivo by catheter-based rat model preparation. The biofilms were examined before and after the treatment using a scanning electron microscope. Results and conclusion The produced nonthermal plasma degenerated and reduced the number of adherent and aggregated viable bacteria.