Abstract Background and Objective: Nowadays, incidence of antibiotic-resistance among pathogenic bacteria has increased due to indiscriminate use of antimicrobial drugs for treatment of diseases, especially urinary tract infections. Medicinal plants are also of great importance as antibacterial agents. Therefore, the aim of this study was to determine the antibacterial effect of ethanolic extract of nettle (Urtica dioica L.) leaves using two methods of disk diffusion and well diffusion. Methods: Ethanolic extract of nettle leaves was prepared by the percolation method. Effect of different concentrations of the extract on Escherichia coli (PTCC1399), Staphylococcus aureus (PTCC 1431), Staphylococcus epidermidis (PTCC 1435) and Staphylococcus saprophyticus (PTCC1440) was evaluated using the disk diffusion and well diffusion methods by measuring diameter of growth inhibition zone. Gentamicin and propylene glycol were used as positive and negative control, respectively. Results: In both methods, especially in the well diffusion, the ethanolic extract of nettle leaves had favorable inhibitory effect on the growth of S. aureus, S. epidermidis and S. saprophyticus. In the well diffusion method, the highest rate of susceptibility to the extract (89%) was related to S. saprophyticus and S. epidermidis. Conclusion: The ethanolic extract of nettle leaf has good inhibitory effect on the growth of S. aureus (especially in the well diffusion method), which confirms the traditional use of this plant for the treatment of urinary tract infections.