Antibacterial biodegradable PLA-based nanofibers loaded with phenolic monoterpenes - thymol, eugenol, carvacrol, and cinnamaldehyde, were prepared by electrospinning. The effect of bioactive molecule on the surface, thermal, morphological, and biological properties has been investigated about the potential pharmaceutical and food processing applications. Fiber diameters ranged from 320nm for PLA fibrous mat up to 480nm for PLA membrane with 6% thymol. All the prepared active nanofibers exhibited hydrophobic surfaces with a slightly decreasing contact angle after the incorporation of phenols. Antimicrobial testing proved a strong efficiency against Escherichia coli and Staphylococcus aureus, depending on the specific type and content of the bioactive compound. A significant biofilm formation reduction of bioactive PLA nanofibers was revealed against tested microorganisms. Modification of PLA fibers with active molecules did not significantly affect the biodegradation kinetics in comparison to PLA samples with their absence. This study demonstrates the high potential of newly developed PLA-based/phenol nanofibrous membranes for use as antibacterial and antifouling systems applicable in wound dressings and food packaging.