Organs‐on‐chip (OoCs) are catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative. OoCs enable the creation of three‐dimensional (3D) tissue microenvironments with physiological and pathological relevance at unparalleled precision and complexity, offering new opportunities to model human diseases and to test the potential therapeutic effect of drugs, while overcoming the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver‐on‐a‐chip model to investigate the effects of two naturally occurring polyphenols, namely quercetin and hydroxytyrosol, on nonalcoholic fatty liver disease (NAFLD) using a high‐content analysis readout methodology. NAFLD is currently the most common form of chronic liver disease; however, its complex pathogenesis is still far from being elucidated, and no definitive treatment has been established so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid‐induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant and lipid‐lowering properties. In conclusion, the findings of the present work could guide novel strategies to contrast the onset and progression of NAFLD. Gori and coworkers developed a microfluidic model of nonalcoholic fatty liver disease (NAFLD) within a liver‐on‐a‐chip platform, to assess the effect of two natural polyphenols (namely, quercetin and hydroxytyrosol) via high‐content analysis. The two polyphenols showed a hepatoprotective activity against free fatty acid overload, thus reducing hepatic steatosis. This work suggests a potential application of quercetin and hydroxytyrosol in the treatment of NAFLD, and underlines the emerging role of organs‐on‐chip in disease modelling.