Human exposure to carbon nanotubes (CNTs) can cause health issues due to their chemical-physical features and biological interactions. These nanostructures cause oxidative stress, also due to endogenous reactive oxygen species (ROS) production, which increases following mitochondrial impairment. The aim of this in vitro study was to assess the health effects, due to mitochondrial dysfunction, caused by a sub-chronic exposure to a non-acutely toxic dose of multi walled CNTs (raw and functionalised). The A549 cells were exposed to multi-walled carbon nanotubes (MWCNTs) (2 µg mL ) for 36 days. Periodically, cellular dehydrogenases, pyruvate dehydrogenase kinase 1 (PDK1), cytochrome c release, permeability transition pore (mPTP) opening, transmembrane potential (Δψ m), apoptotic cells, and intracellular ROS were measured. The results, compared to untreated cells and to positive control formed by cells treated with MWCNTs (20 µg mL ), highlighted the efficiency of homeostasis to counteract ROS overproduction, but a of mitochondrial functionality was not observed. Despite the tendency to restore, the mitochondrial impairment persisted. Overall, the results underlined the tissue damage that can arise following sub-chronic exposure to MWCNTs.