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A biological assay was carried out to evaluate the impact of dietary tryptophan (TRP) in aflatoxin B1‐contaminated diets (AFB1‐D) on performance, blood parameters, immunity, meat quality and microbial populations of intestine in Japanese quails. Six experimental diets were formulated to include two levels of dietary TRP; 2.9 (moderate high: MH‐TRP) and 4.9 g/kg (excess: Ex‐TRP); and three levels of AFB1 (0.0, 2.5, and 5.0 mg/kg). Each experimental diet was fed to the one of the six groups of birds from 7 to 35 days of age in a completely randomized design with 2 × 3 factorial arrangement. Decrease in feed intake, body weight gain and gain:feed in birds fed 5.0 mg/kg AFB1‐D was restored to the control level by 4.9 g TRP/kg of the diet. The hepatic enzymes in blood were elevated in quails fed on AFB1‐D but attenuated by 4.9 g TRP/kg of the diet (Ex‐TRP; p ≤ .01). High serum uric acid in birds challenged with AFB1 significantly decreased by Ex‐TRP (p ≤ .01). The skin thickness to 2,4‐dinitro‐1‐chlorobenzene challenge suppressed by AFB1 but increased by Ex‐TRP diet (p ≤ .02). The AFB1 increased the malondialdehyde in meat, whereas TRP efficiently diminished malondialdehyde production (p ≤ .01). The greatest drip loss and pH in meat were observed in the birds fed 5.0 mg/kg AFB1‐D but Ex‐TRP augmented the adverse effects of AFB1 (p ≤ .01). The Ex‐TRP reduced the total microbial and Escherichia coli counts (p ≤ .01). The adverse effect of AFB1 on ileal Lactic acid bacteria was completely prevented by Ex‐TRP (p ≤ .03). This study showed that tryptophan supplementation could be considered as a powerful nutritional tool to ameliorate the adverse effects of AFB1 in growing quails.