Azacitidine (AZA) is recognized as a vital drug used in the therapy of myelodysplastic syndromes (MDS) due to its beneficial effect on survival and quality of life. Nevertheless, many patients fail to respond to AZA treatment, as prognostic factors still are not identified. The present retrospective analysis included 79 patients with MDS treated with AZA as first-line therapy in a real-life setting. The percentage of patients with good, intermediate, and poor cytogenetics was 46.8%, 11.4%, and 34.2%, respectively. The overall response rate (complete remission [CR], partial remission [PR], and hematological improvement [HI]) was 24%. The CR, PR, and HI rates were 13.9%, 2.5%, and 7.6%, respectively. Stable disease (SD) was documented in 40.5% of patients. The median overall survival (OS) and progression-free survival (PFS) were 17.6 and 14.96 months, respectively. Patients with ORR and SD had a significantly longer median OS (23.8 vs. 5.7 months, p = 0.0005) and PFS (19.8 vs. 3.5 months, p < 0.001) compared to patients who did not respond to AZA. In univariate analysis, only an unfavorable cytogenetic group was a prognostic factor of a lower response rate (p = 0.03). In a multivariate model, older age (p = 0.047), higher IPSS (International Prognostic Scoring System) risk (p = 0.014), and higher IPSS-R cytogenetic risk (p = 0.004) were independent factors of shorter OS. Independent prognostic factors for shorter PFS were age (p = 0.001), IPSS risk (p = 0.02), IPSS cytogenetic risk (p = 0.002), and serum ferritin level (p = 0.008). The safety profile of AZA was predictable and consistent with previous studies. In conclusion, our study confirms the efficacy and safety of AZA in a real-world population and identifies potential biomarkers for response and survival.