Abstract Background: Acute myeloid leukemia (AML) with FLT3 mutations carries a poor prognosis. FLT3 inhibitors, such as midostaurin, combined with standard chemotherapy, have shown improved outcomes for FLT3-mutated AML patients. However, real-world data on their efficacy and safety are limited. This study assesses midostaurin's real-world efficacy, the timing of molecular diagnosis, and the impact of antifungal prophylaxis management. Methods: This retrospective multicenter study was conducted across four Spanish hospitals, including 15 patients with FLT3-mutated AML treated between 2019 and 2024. Data on demographics, clinical characteristics, diagnostic timelines, and treatments were collected and analyzed. Results: The average time to obtain FLT3 mutation results was 13.3 days, exceeding the recommended 8-day window per PETHEMA, NCCN, and ELN guidelines, and delaying midostaurin initiation to a mean of 24.5 days post-diagnosis. Despite these delays, 80% of patients achieved complete remission (CR), with 62.5% attaining negative minimal residual disease (MRD) status post-induction. Antifungal prophylaxis was administered to 60% of patients, primarily with posaconazole (62.5%), without midostaurin dose adjustments. Alternative antifungals (micafungin or fluconazole) were used to avoid CYP3A interactions per PETHEMA recommendations. No significant differences in adverse event rates were observed between antifungal regimens. Conclusion: The real-world use of midostaurin demonstrates efficacy comparable to clinical trials. However, delays in FLT3 mutation testing hinder adherence to treatment initiation guidelines, potentially impacting outcomes. Additionally, the lack of robust evidence regarding interactions between midostaurin and antifungal agents highlights the need for standardized antifungal prophylaxis strategies in FLT3-mutated AML. Addressing these challenges is essential to optimize outcomes in real-world settings. Key words: Acute myeloid leukemia; FLT3 mutation; Midostaurin; Real-world evidence; Antifungal prophylaxis; Minimal residual disease.