Since the global restrictions on antibiotics in poultry systems, there has been a growing demand for natural and sustainable feed additives for disease prevention and poultry nutrition. This study evaluated the effects of incorporating sugarcane bagasse (SCB) lignin into broiler chicken diets. The performance of the chickens, including body weight, feed intake, and mortality, as well as intestinal histomorphometry, and cecum content pH, microbiota, and volatile fatty acids were assessed. In addition, we also aimed to validate an in vitro gastrointestinal tract (GIT) model developed by Carvalho et al. (2023). One hundred and eight 1-day-old Ross 308 chicks were randomly and equally divided into two groups. The first group was fed a basal diet (BD group), while the second group was fed a basal diet supplemented with 1% (w/w) SCB lignin (BD + SCB lignin group) for 36 days. The in vivo conditions of the chicken GIT were replicated in an in vitro model. In the in vivo study, SCB lignin increased cecum acetate and butyrate levels while reducing Bifidobacterium and Enterobacteriaceae, without affecting productivity (body weight, feed intake, and mortality). The in vitro assessment reflected microbiota trends observed in vivo, although without statistical significance. The divergence in organic acid production between the in vivo and in vitro conditions likely resulted from issues with inoculum preparation. This study demonstrates that SCB lignin incorporation positively influences cecal microbiota composition without impacting the animals’ productivity and physiology, suggesting its potential as a functional feed additive. For a more reliable in vitro model, adjustments in inoculum preparation are necessary.