The influence of non-thermal plasma (NTP) treatment on the prevention of antibiotic resistance of microbial biofilms was studied. Staphylococcus epidermidis and Escherichia coli bacteria and a yeast Candida albicans, grown on the surface of Ti-6Al-4V alloy used in the manufacture of prosthetic implants, were employed. Their biofilms were exposed to NTP produced by DC cometary discharge and subsequently treated with antibiotics commonly used for the treatment of infections caused by them: erythromycin (ERY), polymyxin B (PMB), or amphotericin B (AMB), respectively. All biofilms displayed significant reduction of their metabolic activity after NTP exposure, the most sensitive was S. epidermidis. The subsequent action of antibiotics caused significant decrease in the metabolic activity of S. epidermidis and E. coli, but not C. albicans, although the area covered by biofilm decreased in all cases. The combined effect of NTP with antibiotics was thus proved to be a promising strategy in bacterial pathogen treatment.