Graphene Oxide has been proposed as a potential adjuvant to develop improved anti-TB treatment, thanks to its activity in entrapping mycobacteria in the extracellular compartment limiting their entry in macrophages. Indeed, when administered together with linezolid, Graphene Oxide significantly enhanced bacterial killing due to the increased production of Reactive Oxygen Species. In this work, we evaluated Graphene Oxide toxicity and its anti-mycobacterial activity on human peripheral blood mononuclear cells. Our data show that Graphene Oxide, different to what is observed in macrophages, does not support the clearance of Mycobacterium tuberculosis in human immune primary cells, probably due to the toxic effects of the nano-material on monocytes and CD4+ lymphocytes, which we measured by cytometry. These findings highlight the need to test GO and other carbon-based nanomaterials in relevant in vitro models to assess the cytotoxic activity while measuring antimicrobial potential.