Coffee contains human health-related molecules, namely polyphenols that possess a wide range of pharmacological functions, and their intake is associated with reduced colon cancer risk. This study aimed to assess the changes in the anti-inflammatory and antioxidant activity of coffee after simulated gastrointestinal digestion. The evaluation of intracellular reactive oxygen species (ROS) levels in the HT-29 human colon cancer cell line and three in vitro spectrophotometric assays were performed to determine the antioxidant activity of the samples. Characterization of coffee composition was also assessed through a Q-Orbitrap high-resolution mass spectrometry analysis. The results highlighted that the levels of polyphenols in the digested coffee brews were higher than those of the non-digested ones. All assayed samples decreased the levels of intracellular ROS when compared to untreated cells, while digested coffee samples exhibited higher antioxidant capacity and total phenolic content than not-digested coffee samples. Digested coffee samples showed a higher reduction in interleukin-6 levels than the not-digested samples in lipopolysaccharide-stimulated HT-29 cells treated for 48 h and fewer cytotoxic effects in the MTT assay. Overall, our findings suggest that coffee may exert antioxidant and anti-inflammatory properties, and the digestion process may be able to release compounds with higher bioactivity.