<p>Growing concerns over antibiotic resistance and environmental sustainability have increased the demand for natural alternatives to in-feed antibiotics in poultry production. Biochar offers a novel, eco-friendly solution that may improve animal performance while supporting waste valorization. This study investigated the effect of biochar produced from pistachio by-product (PB) on growth, feed efficiency, and gut health in Japanese quails. Birds were assigned to five dietary treatments including a control diet without additives, a diet with 0.05% flumequine, and diets supplemented with 0.35%, 0.65%, or 1% PB biochar for 35 days. Feed intake, weight gain, feed conversion ratio (FCR), carcass traits, nutrient digestibility, intestinal histomorphology, and cecal microbial counts were evaluated. The results showed that final body weight and weight gain significantly increased in birds fed 0.65% biochar compared to the control and flumequine groups (<i>P</i> &lt; 0.05). A trend towards a lower FCR was observed in birds fed 0.65% biochar compared with the control (<i>P</i> = 0.08). Except for the weight of the breast and thigh, none of the carcass traits and internal organs were affected by the treatments (P &gt; 0.05). Preplanned comparisons between control and biochar-treated groups showed that PB biochar significantly increased Lactobacillus populations while reducing E. coli counts in the cecum (<i>P</i> &lt; 0.05). Ileal digestibility of crude protein (CP) and crude fiber (CF) also enhanced with the inclusion of biochar in the diets compared to the control (<i>P</i> &lt; 0.05). Dietary inclusion of PB biochar increased cecal and ileal villus height indicating improved gut morphology (<i>P</i> &lt; 0.05). The optimal inclusion level of PB biochar was found to be 0.65%, which showed significant improvements in growth and digestibility. Overall, this study provides insight into the potential of PB biochar as a promising alternative to antibiotics.</p>