The widespread use of quinolones (QNs) in human and veterinary medicine has resulted in the emergence of QN-resistant bacteria. The most common and widespread type of resistance to FQ is the presence of point mutations in the QRDR region and/or PMQR genes. The aim of the study was to analyze the mechanisms of quinolone resistance among E. coli strains isolated from wild animals in Poland. Out of 42 tested isolates, 39 strains had at least one point mutation in the gyrA gene; however, 76.2% of the isolates had at least three mutations in the GyrA and ParC domains, mainly S83L or D87L in the gyrA gene and S80I in the parC gene. Single isolates had also other mutations: D87Y in the GyrA domain and E84G, A108V, G78C, or S80R in the ParC domain. In 12 strains (28.5%), including three in which no QRDR mutations were confirmed, the presence of single PMQR genes: qnrB, qnrS, and aac(6’)-Ib-cr was also demonstrated. Accumulation of point mutations was associated with high values of MIC of fluoroquinolones and single mutations or the presence of PMQR genes were associated with low-level resistance (MIC from 2 to 8 mg/L). The comparative method (MAS-PCR) confirmed the results of sequencing in most cases (90.5%). The E. coli strains isolated from the wildlife were characterized by the same mechanisms of QNs-resistance as those most often identified among isolates from anthropogenic environments. The obtained results indicate that the level of QNs-resistance is really high and monitoring should be obligatorily extended to the natural environment.