Water environments play an important role in the dissemination of antibiotic-resistant bacteria among humans, animals and agricultural sources. In order to assess the spread of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, we analyzed 279 effluent samples from 21 wastewater treatment plants in Navarra (northern Spain). A total of 185 cefpodoxime-resistant bacteria were isolated on ChromID ESBL agar plates, with high predominance of Escherichia coli among isolated species (73%). ESBL production was determined by different methods, concluding its presence in 86.5% of the isolates by the combination disk test, 75.7% by double-disk synergy test and 73.5% by MicroScan® NM37 automated system. PCR and sequencing analysis showed that the predominant ß-lactamases (bla) genes were blaCTx-M (67.4%) followed by blaTEM (47%), blaSHV (17.4%) and blaOxA (8.3%); furthermore, two or more ß-lactamases genes were found in 34.9% of the isolates. The results demonstrate the high prevalence of ESBL-producing Enterobacteriaceae in effluent water from wastewater treatment plants and confirm the need to optimize current disinfection procedures and to improve management of wastewater in an effort to minimize reservoirs of resistant bacteria. Further studies are needed for examining the presence of these bacteria in other environments and for determining the potential dissemination routes of these resistances as well as their impact on human health.