Background <p>This study aimed to characterize the longitudinal and dynamic changes of gut microbiota in patients with nasopharyngeal carcinoma (NPC) during radiotherapy, to inform the development of strategies for maintaining gut microbiota homeostasis.</p> Methods <p>Thirty-one newly diagnosed NPC patients were recruited from the Department of Radiotherapy, First Affiliated Hospital of Guangxi Medical University, and 31 age-matched healthy controls from the same Hospital Physical Examination Center between August and December 2023. Fecal samples were collected from NPC patients at three key time points, including days 1–3 pre-radiotherapy (T0), days 14–18 of mid-radiotherapy (T1), and days 28–32 at end of radiotherapy (T2). Meanwhile, fecal samples from healthy controls were collected on the morning of their physical examination. 16S rDNA sequencing, combined with bioinformatics and statistical analyses, was used to compare the structural characteristics and dynamic changes of gut microbiota in NPC patients during radiotherapy.</p> Results <p>Alpha diversity analysis showed significantly lower Shannon and Chao1 indices in NPC patients compared with healthy controls. Beta diversity analysis based on the Bray-Curtis distance revealed distinct gut microbial community structures between NPC patients and healthy controls. In this intergroup comparison, a total of 16 core differentially abundant taxa were consistently identified by two complementary analytical methods (LEfSe and ALDEx2). Linear mixed-effects models demonstrated that the Chao1 index at T0 was significantly lower than at T1 and T2 in NPC patients. Following adjustment for multiple confounding variables, mucositis grade was the only factor significantly associated with gut microbiota alpha diversity during radiotherapy. Radiotherapy time points, treatment regimen, and most clinical and demographic variables showed no such association. Beta diversity analyses based on the Bray-Curtis and unweighted Unifrac distances revealed significant compositional and structural differences linked to radiotherapy time points (T0/T1/T2) in the gut microbiota of NPC patients. This temporal variation was abrogated by stratification according to chemotherapy regimen. Beta diversity based on Bray-Curtis distance showed no significant differentiation across time points in either the concurrent chemotherapy (CCRT) alone cohort or the induction chemotherapy plus CCRT cohort. LEfSe and ALDEx2 concordantly identified seven core differentially abundant taxa across all NPC patients in the longitudinal T0 vs. T1 vs. T2 analysis. Stratification by chemotherapy regimen revealed such core taxa in the CCRT alone cohort, whereas no core differentially abundant taxa were detected by either method in the induction chemotherapy plus CCRT cohort.</p> Conclusion <p>Our findings demonstrate that NPC patients exhibit significant gut microbial dysbiosis compared with healthy controls, characterized by reduced alpha diversity and altered genus-level composition. Longitudinal analyses further revealed that radiotherapy is associated with dynamic alterations in gut microbial diversity, composition, and structure in NPC patients. Notably, these temporal shifts in the gut microbiota are strongly stratified by chemotherapy regimen, with pronounced changes observed in patients receiving CCRT alone but a stable microbial profile in those receiving induction chemotherapy followed by CCRT. Collectively, these results highlight the profound impact of oncological treatment on the gut microbiome in NPC patients and identify treatment regimen as a critical modifier of microbial dynamics during radiotherapy.</p>