A prototype that generates non-thermal plasma directly and solely in a sealed bag has been developed (Patent PTC/FR2011/05219) and tested on Pseudomonas aeruginosa biofilms so as to see the bactericidal power of such non-thermal plasma. We assessed the effectiveness of two different plasma produced from gas mixtures of oxygen and nitrogen (5% O₂–95% N₂ or 15% O₂–85% N₂) on P. aeruginosa biofilms cultured on disks made of a hydroxyapatite-coated titanium alloy. We consequently obtained a bactericidal effect after a 60-min treatment on biofilms (5.8 log reduction) regardless of the gas mixtures. The relative effectiveness of plasma on bacterial biofilms could be explained by the thickness of biofilms and the difficulties to destroy the bacteria in the deeper layers that featured different physiological states. Another hypothesis dealt with the plasma partial diffusion in the hydroxyapatite porosities that coat the titanium alloy disks.