Abstract The optimal sequence for administration of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for treating non-small cell lung cancer (NSCLC) is still unclear. This study aimed to evaluate the efficacy of sequential afatinib and osimertinib treatment in patients with NSCLC harboring EGFR mutations. Electronic records of patients with EGFR-mutated NSCLC, who were administered afatinib and osimertinib (group A) or other chemotherapy (group B) between October 2014 and October 2019, across 16 hospitals in South Korea were reviewed. The primary outcome, time on treatment (TOT), secondary outcome, and overall survival (OS) were estimated using the Kaplan-Meier method and log-rank test. Multivariate analyses were performed using the Cox proportional hazards model. Of the 737 patients who received frontline afatinib treatment, 360 with complete records were selected (group A:154, group B: 206). The median TOT was 33.9 months (95% confidence interval [CI]: 24.5−43.3) in group A and 21.3 months (95% CI: 19.4−23.1) in group B. The median TOT with afatinib was 12.9 months (95% CI: 11.8−14.0) overall, and 15.2 months (95% CI: 13.2−17.1) in group A. The 2- and 3-year survival rates were 86.0% and 69.3% in group A and 75.9% and 55.3% in group B, respectively. Sequential afatinib and osimertinib treatment resulted in better survival rates than treatment with afatinib followed by other chemotherapies. Therefore, this sequential treatment strategy may offer clinical benefits to patients with EGFR-mutated NSCLC.