The objective of this study was to determine the drug resistance profile and the presence of genes that cause the ESBL mechanism in Escherichia coli isolated from brine that circulates in a closed-cycle graduation tower. A disk diffusion method was employed to evaluate antibiotic resistance and a polymerase chain reaction (PCR) technique with specific primers was used to detect genes that cause the production of extended-spectrum β-lactamases (ESBLs). The total number of E. coli strains isolated was 40. The highest observed resistance levels were noted for ampicillin, amoxicillin/clavulanic acid, and ticarcillin. Most strains were resistant to 2 antibiotics, but one strain was found to be resistant to up to 8 antibiotics simultaneously. No MDR (multidrug-resistant) strains were observed. A total of 39 isolates were subjected to gene testing, and at least one gene conditioning the extended-spectrum β-lactamase (ESBL) mechanism was identified. The most prevalent genes responsible for β-lactamase production were blaTEM (87.5%) and blaCTXM-9 (70%). The brine feeding the graduation tower was found to be a reservoir of drug-resistant E. coli strains, which represent a potential threat to public health. The presence of drug-resistant E. coli in the brine water may be a health risk to those inhaling in the graduation tower, which casts doubt on the health-promoting properties of such treatments. The results suggest the need for further research to observe the propagation of drug resistance in E. coli.