Responses to stocking density (SD), dietary biotin concentration and litter condition were evaluated on 2016 Ross 308 male broilers in the fattening period (day 22-day 42). The birds were placed in 48 pens with either dry or wet litter to simulate the final stocking density of 30 kg (12 broilers/m ; normal stocking density, NSD) and 40 kg (16 broilers/m ; high stocking density, HSD) of body weight (BW)/m floor space. A corn-soybean meal-based diet was supplemented with biotin to provide a normal (NB; 155 μg/kg) or high (HB, 1521 μg/kg) level of dietary biotin. There were six repetitions per treatment. The inappropriate moisture content of litter associated with HSD was avoided (p < 0.05) by good management (SD difference: dry litter, 6.65% vs. wet litter, 13.23%; 42 days), which made it advantageous (p < 0.01) for footpad (SD difference: dry litter, 0.118 vs. wet litter, 0.312; weekly average value) and hock health (SD difference: dry litter, 0.090 vs. wet litter, 0.303; weekly average value) of HSD birds, but not (p > 0.05) for growth and processing yield. In HSD, the biotin effect (gains, FCR) was significantly higher (p < 0.01) than in NSD. The similar response of HSD birds to supplemental biotin was observed (p < 0.05) for lesion scores of footpad and hock in particularly finishing chickens, and a significant interaction (p < 0.01) among stocking density, biotin supplementation and litter condition existed from 35 to 42 days of age. Taken together, increasing dietary biotin improves the performance and well-being of broiler chickens stocked at high densities in litter-independent and litter-dependent manners respectively.