Osteosarcoma (OS) is a rare malignant primary bone tumours characterized by a high genetic and cell composition heterogeneity. Unfortunately, despite the use of drug combinations and the recent development of immunotherapies, the overall survival has not improved in the last four decades. Due to the key role of the tumour microenvironment in the pathogenesis of OS, a better understanding of its microenvironment is mandatory to develop new therapeutic approaches. From retrospective biological cohorts of OS, we analysed by immunohistochemistry the presence of lipopolysaccharide (LPS)-binding protein (LBP) in diagnostic biopsies with local disease and compared their level of infiltration to patients suffering from metastatic status. LBP is considered as a marker of LPS exposure and can indirectly reflect the presence of Gram-negative microbiota. LBP were detected in the cytoplasm of OS cells as well as in tumour-associated macrophage. Tumour samples of patients with local disease were significantly enriched in LBP compared to tumour tissues of patients with metastatic status. Lung metastatic tissues showed similar level of LBP compared to paired primary tumours. Overall, this study strongly suggests the presence of Gramnegative bacteria in OS tissues and demonstrated their significant differential level according the metastatic status. This tumour-associated microbiome may help in the conceptualisation of new therapeutic approach to trigger efficient therapeutic responses against cancer.