Background Clinical guidelines (CG) are used to reduce variability in practice when the scientific evidence is sparse or when multiple therapies are available. The development and implementation of evidence-based CG is intended to organize and provide the best available evidence to support clinical decision making in order to improve quality of care. Upper respiratory tract infections (URTI) are the leading cause of misuse of antibiotics and a CG may reduce the unnecessary antibiotic prescription. Methods The aim of this quasi-experimental, before-after study was to analyze the short- and long-term effects of the implementation of a CG to decrease the rate of antibiotic prescription in URTI cases in the emergency department of a third level private hospital in Quito, Ecuador. The study included 444 patients with a main diagnosis of URTI. They were distributed in three groups: a baseline cohort 2011 (n = 114), a first post-implementation cohort 2011 (n = 114), and a later post-implementation cohort 2018 (n = 216). The implementation strategy consisted of five key steps: acceptance of the need for implementation of the CG, dissemination of the CG, an educational campaign, constant feedback, and sustainability of the strategy through continuous training. Results The results of this study show a 42.90% of antibiotic prescription rate before the CG implementation. After the implementation of the CG, the prescription rate of antibiotics was significantly reduced by 24.5% (42.9% vs 18.4%, p<0.0001) and the appropriate antibiotic prescription rate was significantly increased by 44.2% (22.4% vs 66.6%, p<0.0001) in the first post-implementation cohort 2011. There was not a significant difference in antibiotic prescription rate and appropriate antibiotic prescription rate between two post-implementation cohorts: 18.4% vs 25.9% (p = 0.125) and 66.6% vs 50% (p = 0.191), respectively. Conclusions The implementation of CGs decreases the rate of antibiotic prescription in URTI cases. The results are remarkable after early implementation, but the effect persists over time. The emphasis must shift from guideline development to strategy implementation.