Environmental heat stress causes significant economic loss in the poultry industry. Therefore, interest has increased in using feed additives to reduce the negative impacts of heat stress on the chickens and improve production performance. This study aimed to assess the effect of supplementing with Nigella sativa nanoparticles (Nano-NS) as an anti-stress and growth promoter in broiler diets under hot climatic conditions. A total of 375 male one-day-old Ross 308 chicks were randomly divided into a control group and four treatment groups (75 chicks/group). The first group fed a basal diet without additives, the second group fed a basal diet supplemented with avilamycin at 50 mg/kg, and the other groups fed a basal diet supplemented with 30, 40, and 50 mg/kg Nano-NS, respectively. Despite that feed intake was not affected, feed conversion ratio, body weight gain, and crude protein digestibility improved in broilers fed Nano-NS (P < 0.05) compared with avilamycin and the control groups. Adding Nano-NS led to an increase in the dressing percentage and the relative weight of the bursa of Fabricius and thymus. Serum high-density lipoprotein levels increased while total cholesterol and low-density lipoprotein concentrations decreased (P < 0.05) in broilers fed Nano-NS compared with control groups. Furthermore, Nano-NS supplementation significantly increased (P < 0.05) serum immunoglobulin (IgG and IgA), and superoxide dismutase (SOD) levels, while decreasing malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) concentration. Moreover, there was a significant increase in the Lactobacillus population and a decrease (P < 0.05) in the E. coli and C. perfringens population in chicks fed Nano-NS. In the intestinal tissues, mucin 2 (MUC2) gene expression increased in chickens fed 50 mg/kg Nano-NS compared to other groups. It is concluded that adding Nano-NS (up to 50 mg/kg) reduced the negative effects of heat stress via enhancing growth performance, immune responses, and antioxidant status, modulating the microbial community structure, and increasing the expression of the MUC2 gene in broilers under high ambient temperature.