The objective of this work was to evaluate antibacterial and antibiofilm activity of Eucalyptus camaldulensis EO-loaded PLA nanofibers. Different concentrations of E. camaldulensis EO (7.5, 10 and 15%) were incorporated into PLA nanofibers via an electrospinning technique. The nanofibers are characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and dynamic mechanical analysis (DMA). Antibacterial activity of essential oil-loaded PLA nanofibers against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis were investigated by disc diffusion method. The antibiofilm activity of essential oil-loaded PLA nanofibers was studied against Pseudomonas aeruginosa. The results obtained from the biofilm formation test revealed that PLA essential oil inhibited the Pseudomonas aeruginosa biofilm by 9.3%, 12.8% and 12.6%, respectively. Drug releasing behavior of essential oil-loaded PLA nanofibers was investigated by UV-Visible spectroscopy. According to releasing results, as the amounts of Eucalyptus camaldulensis essential oil in PLA nanofibers increased, the releasing decreased. The essential oil-loaded PLA nanofiber successfully delivered about 160 h.