Cell stimulation using piezoelectric polymers, which is known as piezostimulation, is an innovative approach for designing antimicrobial protection. As an antibiotic-free and inorganic nanoparticle-free approach, it uses physical stimuli to target bacterial cells in a non-specific manner, which may be of great importance, particularly in the context of avoiding resistant bacterial strains. In this study, we prepared fully organic piezoelectric biodegradable films composed of poly-L-lactide (PLLA) and demonstrated their antimicrobial effect on S. epidermidis as a model of Gram-positive and E. coli as a model of Gram-negative bacteria. The PLLA films were either smooth and fabricated using simple melt- drawing or nanotextured, as self-standing nanotubes formed using the template-assisted method. The morphological differences between nanotextured and smooth films resulted in a larger surface area and better surface contact in nanotextured films, together with improved structural properties and better crystallinity, which were the main reasons for their better piezoelectric properties, and consequently stronger bactericidal effect. The comparison between the nanotextured surfaces with and without piezoelectric nature excluded the main role of morphology and directly confirmed piezoelectricity as the main reason for the observed antimicrobial affect. We also confirmed that piezo-stimulation using the antibacterial nanotextured film could damage the bacterial membrane as the main mechanism of action, while the contribution of pH changes and ROS generation was negligible. More importantly, the effect was selective toward the bacterial membrane and the same damage was not observed in human red blood cells, making the therapeutic use of these films possible.