Jackfruit seed flour can be used as a cocoa aroma replacer with similar technological properties. The purpose of this study was to investigate the in vivo toxicity and in vitro antioxidant activity of fermented jackfruit seed flour (Fjs) and non-alkaline cocoa powder (Nac). Fjs and Nac extracts were produced and submitted to in vitro gastrointestinal digestion producing digested fractions named Fjs-D and Nac-D, respectively. Nac-E showed over two-fold higher oxygen radical absorbance capacity (ORAC) than Fjs-E. However, after simulated gastrointestinal digestion (in vitro), there were no significant differences between Nac-D and Fjs-D (p < 0.01). Similarly, the cellular antioxidant activity (CAA) of Nac-D and Fjs-D was not significantly different (p < 0.01). The anti-inflammatory assay in transgenic RAW 264.7 murine macrophages showed that Fjs-E did not affect cell viability up to 300 μg mL-1 (p > 0.05) and reduced by 15% the release of TNF-α (p < 0.05). Fjs-D did not affect cell viability up to 300 μg mL-1 (p > 0.05) and showed 58% reduction of NF-κB activation (p < 0.05), with no effects on TNF-α levels. Treatment with Nac-E up to 300 μg mL-1 did not decrease cell viability (p > 0.05) and reduced the release of TNF-α levels by 34% and 66% at 100 and 300 μg mL-1 , respectively (p < 0.05). Nac-D did not reduce the NF-κB activation or TNF-α levels at any tested concentration. Collectively, these findings indicate that Fjs is safe and promising functional ingredient with biological activities even after gastrointestinal digestion. This article is protected by copyright. All rights reserved.