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 BR-CNFs, the toxicity in vitro and in vivo was evaluated on Caco-2 intestinal cells and mice, respectively. The results showed that CNFs obtained from banana rachis 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 in vivo cytotoxicity assays indicated that mice showed no sign of pathogenicity or lesions in the liver, kidney, or small intestine when treated with a single dose of BR-CNFs obtained from the banana rachis. Moreover, when the mice were treated daily for a month with BR-CNFs was not observed any hyperplasia or hypertrophy of the organs evaluated. Additionally, biochemical parameters such as blood chemistry, creatinine, liver enzymes, and renal function showed that the BR-CNFs do not represent organ damage. Overall, this study shows that BR-CNFs are not cytotoxic and genotoxic. In conclusion, these studies are essential to guarantee the safety of this product with high added value in the food industry.