Cellulose nanofibrils from the banana rachis are a good alternative as packaging materials, food packaging, stabilizing agents, and functional food ingredients. To address the potential effects of ingested banana rachis cellulose nanofibrils (BR-CNFs), their toxicity and was evaluated using Caco-2 intestinal cells and mice, respectively. The results showed that BR-CNFs did not cause cytotoxic effects at the concentrations evaluated on Caco-2 cells. In addition to cytotoxicity tests, genotoxicity assays using comet assay indicated that Caco-2 cells showed no DNA damage at the concentrations of CNFs tested. Finally, acute cytotoxicity assays indicated that mice showed no sign of pathogenesis or lesions in the liver, kidney, or small intestine when treated with a single dose of BR-CNFs. Moreover, when the mice were treated daily for a month with BR-CNFs no hyperplasia or hypertrophy was observed in any of the organs evaluated. Additionally, biochemical parameters such as blood chemistry, creatinine, liver enzymes, and renal function showed that the BR-CNFs do not cause organ damage. Overall, this study shows that BR-CNFs are neither cytotoxic nor genotoxic. In conclusion, these studies are essential to guarantee the safety of this high value-added product in the food industry.