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Background: This is the first prospective clinical study to evaluate the impact of prebiotic and probiotic use on acute radiation-induced pelvic side effects and associated changes in serum cytokine levels in patients with pelvic malignancies. Methods: This multicenter prospective study included 80 patients with pelvic cancers treated at two centers between 2021 and 2023. Patients were divided into three groups: probiotic + prebiotic, prebiotic only, and nonsupplemented. Serum cytokine levels (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6) and radiation-related side effect scores were assessed at the beginning of pelvic radiotherapy (RT) and again at the fifth week. Based on data normality, the Wilcoxon signed-rank test was used for statistical analysis. Results: According to side effect scores, in the prebiotic-only group, a modest yet statistically significant increase in fecal incontinence (FI; p=0.040) and blood and mucus in the stool (BMS; p=0.049) was observed. Conversely, the probiotic + prebiotic group showed a significant increase in dysuria (p=0.001) and impotence (p=0.016). IL-6 levels significantly increased in the prebiotic-only group (p=0.035), while TNF-α levels rose significantly in the group without any nutritional support (p=0.025). No significant increase in cytokine levels or intestinal side effects was observed in the combined probiotic + prebiotic group (p  > 0.05 for all comparisons). Conclusions: The absence of a rise in cytokine levels and intestinal toxicity in the probiotic + prebiotic group suggests that probiotics may play a role in mitigating radiation-induced intestinal side effects and inflammatory responses.