Background: Clostridioides difficile infection (CDI) is a frequent complication after hematopoietic stem cell transplantation, but data on its risk factors after autologous transplantation (autoHSCT) are limited. Objective: The aim of this study was to analyze the incidence of CDI after autoHSCT and its association with the underlying diagnosis, the conditioning regimen and fluoroquinolone prophylaxis (FQP). Material and methods: We conducted a retrospective analysis of 113 consecutive autoHSCT procedures performed in a single center between September 2020 and November 2023. Patients with multiple myeloma received melphalan conditioning and patients with lymphoma received BeEAM (bendamustine, etoposide, cytarabine, melphalan). Ciprofloxacin prophylaxis was given in 83 procedures and no antibacterial prophylaxis in 30. Groups were compared using nonparametric tests, and an exploratory logistic regression model with Firth correction was adjusted for the year of transplantation. Results: CDI was diagnosed after 26 of 113 procedures (23.0%). CDI was more frequent in lymphoma patients conditioned with BeEAM than in myeloma patients conditioned with melphalan (44.4% vs. 18.9%; p = 0.030), and this difference remained after adjustment for FQP and year of transplantation (OR = 4.25; 95% CI 1.37-13.22). CDI was less frequent after FQP than without prophylaxis (18.1% vs. 36.7%; p = 0.046). When only toxin-positive cases were counted, the difference between lymphoma and myeloma remained (41.2% vs. 14.4%; p = 0.017), whereas the difference related to FQP was not significant (15.0% vs. 29.6%; p = 0.150). No CDI-related deaths occurred. Conclusions: CDI was common after autoHSCT, especially in lymphoma patients conditioned with BeEAM. The association between FQP and CDI was not consistent and was probably influenced by changes in clinical practice over time. These results should be confirmed in larger, multicenter cohorts.