Abstract Microorganisms are predominantly organized in biofilms, where cells live in dense communities and are more resistant to external stresses compared to their planktonic counterparts. With in vitro experiments, the susceptibility of Candida albicans biofilms to a non-thermal plasma treatment (plasma source kINP®09), in terms of growth, survival, and cell viability was investigated. Behind that background, the C. albicans strain SC5314 (ATCC® MYA-2876™) was plasma treated for different time periods (30 s, 60 s, 120 s, 180 s, 300 s). The results of experiments embracing colony forming units, fluorescence LIVE/DEAD assays, and XTT assays revealed a negative influence of the plasma treatment on the proliferation ability, vitality, and the metabolism of C. albicans biofilms, respectively. Morphological analysis of plasma-treated biofilms using atomic force microscopy supported the indications for lethal plasma effects concomitant with membrane disruptions and the loss of intracellular fluid. Controversial to other publications, fluorescence- and confocal laser scanning-microscopic inspection of plasma-treated biofilms indicated, that an inactivation of cells mainly appeared on the bottom side of the biofilms. If this inactivation leads to a detachment of the biofilms from the overgrown surface, it might offer completely new approaches in the plasma treatment of biofilms. Because of its biochemical-mechanical mode of action, resistances of microbial cells against plasma are unknown at this state of research.