Multi-Walled Carbon Nanotubes (MWCNT) have been functionalized with rutin through three steps (i. reaction step; ii. purification step; iii. drying step) and their physicochemical properties investigated with respect to morphological structure, thermal analysis, Fourier Transform Infrared Spectroscopy (FTIR), and cytotoxicity. The molecular docking suggested the rutin-functionalized MWCNT occurred by hydrogen bonds, which was confirmed by FTIR assays, corroborating the results obtained by thermal analyses. A tubular shape, arranged in a three-dimensional structure, could be observed. Mild cytotoxicity observed in 3T3 fibroblasts suggested a dose–effect relationship after exposure. These findings suggest the formation of aggregates of filamentous structures on the cells favoring the cell penetration.