Cellulose nanoparticles from a vegetable source (cellulose fiber) have been evaluated for future use as reinforcement of polymeric matrixes (e.g., biodegradable films). Cellulose nanoparticles have numerous advantages: they are inexpensive and biodegradable, and they originate from renewable sources. Here, cellulose nanofibers (CNFs) were isolated from banana peel by chemical (alkaline treatment and bleaching followed by acid hydrolysis with 0.1, 1, or 10% (v/v) H2SO4) and mechanical (high pressure homogenizer) treatments. Atomic Force Microscopy (AFM) analysis showed all treatments effectively isolated banana fibers at the nanometer scale (average diameter of 3.72 nm). CNFs displayed ζ-potential values ranging from −37.60 to −67.37 mV, which prevented their aggregation. CNFs had high crystallinity values, from 63.1 to 66.4%, which indicated they could be good reinforcing agents. FTIR results confirmed that the chemical and mechanical treatments removed the amorphous fractions. Regarding cytotoxicity, low CNF concentrations (50–500 μg/mL) did not cause cell death, but CNFs at concentrations above 1000 μg/mL significantly decreased cell viability. The use of different sulfuric acid concentrations provided more detailed knowledge of the treatment methods and CNF features, which could help to improve the CNF production process. The combination of chemical and mechanical treatments proved to be an efficient strategy to prepare CNFs from banana peels as a potential reinforcing agent of polymeric matrixes (e.g., food packaging). Display omitted •CNFs were isolated from banana peel using acid and mechanical treatments.•CNFs showed no cytotoxic effect against Caco-2 at concentrations lower than 500 μg/mL.•Banana peel CNFs have potential application as reinforcing agent in polymeric matrices.