Objective To explore the differences in gut microbiota diversity and structural characteristics among patients with periprosthetic joint infection (PJI), aseptic failure (AF), and osteoarthritis (OA), and to analyze the association between gut microbiota dysbiosis and the occurrence of PJI, thereby providing a new theoretical basis for elucidating the pathogenesis and treatment strategies of PJI in clinical practice. Methods The study enrolled patients with PJI and AF admitted between February 2024 and December 2024, as well as OA patients admitted in February 2024. A total of 52 PJI patients, 19 AF patients, and 29 OA patients who met the selection criteria were included in the analysis. Significant differences were observed among the three groups in terms of gender, age, surgical site, preoperative C-reactive protein levels, and erythrocyte sedimentation rate (P<0.05), while no significant difference was found in American Society of Anesthesiologists (ASA) classification and body mass index (P>0.05). Among the PJI patients, infection staging was as follows: 9 cases in the acute phase, 28 cases in the delayed phase, and 15 cases in the chronic phase; 23 cases were accompanied by sinus tract formation. Fecal samples were collected at different time points: for the PJI group, samples were obtained preoperatively and on postoperative days (7±1) and (14±1); for the AF group, preoperatively and on postoperative day (7±1); and for the OA group, preoperatively only. Metagenomics next-generation sequencing were employed to analyze gut microbiota α-diversity indices (ACE index, Chao1 index, Shannon index, Simpson index, and observed_species index) and differential bacterial genera (screened using the LEfSe algorithm). Results Analysis of gut microbiota diversity showed that the preoperative α-diversity indices (ACE index, Chao1 index, Shannon index, Simpson index, and observed_species index) in the PJI group were significantly lower than those in AF group and OA group (P<0.05). Compared with the AF group on postoperative day (7±1), the α-diversity indices in the PJI group on postoperative day (7±1) were lower, but the difference was not significant (P>0.05); by postoperative day (14±1), these indices further decreased, and the difference was significant (P<0.05). In the PJI group, no significant difference was observed in any of the indices across different time points postoperatively (P>0.05). Analysis of gut microbiota structural characteristics revealed that the PJI group exhibited characteristic dysbiosis both before and after operation. Preoperatively, the PJI group was characterized by enrichment of Pseudomonadota (relative abundance 13.19%), Enterobacteriaceae (Escherichia 3.26%, Klebsiella 1.90%), and opportunistic pathogens such as Enterococcus faecium (0.43%), while the relative abundances of Firmicutes (51.83%) and Bifidobacterium (0.24%) decreased. Postoperatively, the α-diversity in the PJI group further declined, with increased relative abundances of Escherichia and Klebsiella, and the relative abundance of Firmicutes decreased to 40.24%. LEfSe analysis of preoperative gut microbiota composition between the PJI group and AF group indicated that the AF group was predominated by Firmicutes, Bifidobacterium, and Roseburia preoperatively, with greater postoperative microbial stability compared to the PJI group. Conclusion Patients with PJI exhibited a gut microbiota profile characterized by reduced diversity and enrichment of opportunistic pathogens. Postoperative antibiotic treatment further aggravated this dysbiosis, providing new clinical insights into the role of gut microbiota imbalance in the pathogenesis and progression of PJI.