The Latin American region faces a significant challenge with high levels of antimicrobial resistance among important Gram-negative organisms, including E. coli. In recent years, extended-spectrum β-lactamases (ESBLs) have increased in type and frequency, carbapenemases have emerged, and multidrug-resistant E. coli has spread across the American continent. It is also important to recognize the worldwide spread of the E. coli ST131 clones and subclones with fluoroquinolone and ESBL resistance, and in some instances, resistant to carbapemens and aminoglycosides. Resistance in E. coli is generated by positive selection through single point mutations, which is often the case for fluoroquinolone resistance, or by acquisition of mobile genetic elements, which has been the case for broad spectrum penicillins and third-generation cephalosporins. Successful resistance clones may continue to evolve into unique subclones and spread worldwide as observed with ST131 subclones. The frequency of E. coli antimicrobial resistance varies by geographic area and it is not possible to establish universal guidelines on the use of antibiotics. It is critical that every local healthcare institution establishes an antimicrobial stewardship program to promote the proper use of antibiotics, restricting their use, audit clinical use in real-time, and provide feedback to the treating clinician. Implementation of antimicrobial stewardship programs is the best way to optimize clinical outcomes, decrease further antibiotics resistance, and limit healthcare cost associated with management of multidrug-resistant E. coli infections.