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Aims Granulomatous lobular mastitis (GLM) is a rare, chronic, benign inflammatory disease of the breast with an unclear aetiology. This study aimed to characterise the microbial features of GLM using metagenomic next-generation sequencing (mNGS) and to provide potentially relevant microbial clues for clinical evaluation.Methods Twenty fresh lesion tissue samples were collected from 15 female patients with GLM, including one representative sample per patient and five additional deep tissue samples. Clinical data collection, mNGS, bioinformatics analysis and data interpretation were performed to characterise the microbial profiles of GLM lesions.Results In this study, all patients presented with palpable breast masses, breast pain and abscess formation. More than half showed increased white blood cell counts, neutrophil percentages, C reactive protein levels and erythrocyte sedimentation rates together with decreased lymphocyte percentages. Based on genus-level filtering, mNGS identified 16 bacterial genera, 14 fungal genera and 3 viral genera, revealing a complex but bacteria-dominated microbial profile. The most frequently detected bacterial genera were Corynebacterium, Cutibacterium, Acinetobacter, Staphylococcus and Hathewaya, with marked interpatient variation in relative abundance, while fungal profiles were relatively more concentrated. In five patients with both superficial and deep tissue samples, microbial profiles differed across sampling depths, particularly for bacterial composition.Conclusions mNGS revealed a complex, bacteria-dominated microbial profile in GLM lesions and indicated that sampling depth may influence the detected microbial profiles. These findings may provide useful clues for clinical evaluation, but the pathogenic significance of these micro-organisms remains to be elucidated.