The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in Escherichia coli from neonatal intensive care units (NICUs) in Poland. The study was conducted on 80 E. coli isolates from different types of infections collected between 2009 and 2012. Six (5%) isolates were not susceptible to ciprofloxacin, 16% to ofloxacin, and 6.2% to levofloxacin. Among the 80 isolates, 27.5% carried at least one PMQR determinant (n = 22). qnrB was found in 8.8% of isolates (7/80), and qnrS was found in 2.5% (2/80). No isolates carrying qnrA, qnrD, qnrC, qepA, or oqxAB were found. The prevalence of aac(6')-Ib-cr was 27.5%, and 54.5% of the isolates contained aac(6')-Ib-cr and blaCTX-M. qnr-positive strains were more likely to produce extended-spectrum β-lactamases (ESBLs) than qnr-negative strains, which may suggest an association between qnr and ESBLs (88.9% vs. 17%, p = 0.0003). PMQR-positive isolates had significantly higher ciprofloxacin MIC50 values (28.8-fold higher when comparing the MIC50 values) than the PMQR-negative strains (0.23 vs. 0.008 mg/L), regardless of the presence of quinolone resistance-determining region mutations. Five of the nine isolates, for which conjugation was performed successfully, harbored plasmids that contained CTX-M together with qnr and aac(6')-Ib-cr. Our data suggest that the number of qnr genes detected in E. coli from newborns may be related to the selection of qnr through antimicrobial exposure. Even if fluoroquinolones are not commonly used in the NICU, E. coli isolates may carry PMQR. The high prevalence of PMQR is of serious concern, as it may be horizontally transferred to other pathogenic bacteria.