The aim of this study was to evaluate the impact of multi-walled carbon nanotubes (MWCNTs with diameter in the range of 10–30 nm) before and after chemical surface functionalisation on macrophages response. The study has shown that the detailed analysis of the physicochemical properties of this particular form of carbon nanomaterial is a crucial issue to interpret properly its impact on the cellular response. Effects of carbon nanotubes (CNTs) characteristics, including purity, dispersity, chemistry and dimension upon the nature of the cell environment–material interaction were investigated. Various techniques involving electron microscopy (SEM, TEM), infrared spectroscopy (FTIR), inductively coupled plasma optical emission spectrometry, X-ray photoelectron spectroscopy have been employed to evaluate the physicochemical properties of the materials. The results demonstrate that the way of CNT preparation prior to biological tests has a fundamental impact on their behavior, cell viability and the nature of cell–nanotube interaction. Chemical functionalisation of CNTs in an acidic ambient (MWCNT-Fs) facilitates interaction with cells by two possible mechanisms, namely, endocytosis/phagocytosis and by energy-independent passive process. The results indicate that MWCNT-F in macrophages may decrease the cell proliferation process by interfering with the mitotic apparatus without negative consequences on cell viability. On the contrary, the as-prepared MWCNTs, without any surface treatment produce the least reduction in cell proliferation with reference to control, and the viability of cells exposed to this sample was substantially reduced with respect to control. A possible explanation of such a phenomenon is the presence of MWCNT’s agglomerates surrounded by numerous cells releasing toxic substances.