We examined Enterococcus faecalis strains clinically isolated from 100 patients with urinary tract infections (UTIs) for their susceptibility to levofloxacin (LVX) by measuring the MIC and investigated amino acid mutations by direct DNA sequencing, which were then correlated with LVX resistance. Next, we studied risk factors for LVX resistance, such as age, gender, and previous fluoroquinolone use, and investigated the statistical correlation of these risk factors with each amino acid mutation and LVX resistance. Of the 100 isolates tested, 14 isolates showed LVX resistance and all of these isolates had amino acid mutations. We demonstrated that 2 out of 4 mutations (Ser83-to-Ile in gyrA and Ser80-to-Ile in parC) had a significant correlation with LVX resistance. There was a significant relationship between isolates with 2 or 3 amino acid mutations and LVX resistance. In addition, we found a significant correlation between the previous use of fluoroquinolones and LVX resistance or the presence of mutations and also demonstrated that previous use of other types of antibiotics was significantly related to the presence of mutations by multivariate analysis. In conclusion, we found significant correlation between amino acid mutations in E. faecalis, LVX resistance, and risk factors such as previous use of fluoroquinolones.