<p dir="ltr">Tuberculosis remains a global health challenge, ranking as the second leading cause of mortality worldwide in 2022. The resilience of <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis, is enhanced by the high expression of efflux pumps that confer antibiotic tolerance and the formation of biofilms that confer resistance to antibiotics. Carbon nanomaterials (CNMs) exhibit a broad-spectrum of antibacterial efficacy, making them promising candidates for combating drug-resistant bacterial strains. The effects of the novel carbon allotropes called fullertubes (C₉₀) on any living cell have not been studied. In our study, we employed <i>Mycobacterium smegmatis</i> as a model organism for <i>M. tuberculosis</i> and exposed it to fullertubes and fullerenes. We explored the impact of these CNMs on efflux activity and biofilm formation through biochemical assays like ethidium bromide transport assay and crystal violet assay. We also investigated their impact on lipid biosynthesis associated with log-phase growth and biofilm formation using metabolic radiolabeling studies. We also investigated the effects of the efflux pump inhibitors (EPIs) piperine, berberine, 1-(1-naphthylmethyl)-piperazine and thioridazine on efflux activity, biofilm formation, and lipid biosynthesis associated with log-phase growth and biofilm formation in <i>M. smegmatis.</i> We utilized metabolic radiolabeling methods using ¹⁴C-palmitic acid and ¹⁴C-acetic acid which are precursors of lipid biosynthesis and analyzed the lipids by silica-thin layer chromatography and autoradiography. Our studies revealed that CNMs do not influence efflux activity. However, efflux pump inhibitors effectively block efflux activity in <i>M. smegmatis</i>. Biofilm formation was decreased by CNMs and EPIs. In biofilm cells, fullertubes increased the incorporation of radiolabeled ¹⁴C-palmitic acid into glycopeptidolipids on the cell surface as well as inside the cell. Piperine and berberine affected the incorporation of the radiolabels into lipids such as trehalose monomycolate, phosphatidylethanolamine and cardiolipin in planktonic and biofilm cells. Our study provides insights into the diverse effects of CNMs and efflux pump inhibitors on <i>M. smegmatis</i>.</p>