Simple SummaryHeat stress is a serious environmental problem, challenging poultry health and welfare globally, especially during summer season. The breeding program of faster-growing broiler chickens affects their biological homeostasis, causing structural and functional damage to the brain, leading to mental disorders. Improvement in the gut microbiota with synbiotic dietary supplements has become a useful biotherapeutic method for treating various diseases, including neuroinflammation-induced mental illness and memory damage. Therefore, this study aimed to assess the effect of a dietary synbiotic supplement on fear response and memory assessment in heat-stressed broiler chickens. We used 360 1-day-old broiler chicks and assigned them to one of three dietary treatments: a regular diet mixed with a synbiotic containing a probiotic (Enterococcus faecium, Pediococcus acidilactici, Bifidobacterium animalis, and Lactobacillus reuteri) and a prebiotic (fructooligosaccharides) at 0, 0.5, and 1.0 g/kg. Object memory, touch, novel object, isolation, and tonic immobility tests were conducted at relative days of age. At 42 day, blood was collected for detecting corticosterone and tryptophan concentrations and examining heterophile/lymphocyte ratios. The data suggest that the synbiotic-reduced heat-stress responses and related emotional disorder may be mainly caused by increasing the activation of the serotonergic system via the microbiota–gut–brain axis. The aim of this study was to evaluate the effect of a synbiotic containing a probiotic (Enterococcus faecium, Pediococcus acidilactici, Bifidobacterium animalis, and Lactobacillus reuteri) and a prebiotic (fructooligosaccharides) on fear response, memory assessment, and selected stress indicators in broilers subjected to heat stress. A total of 360 1-day-old Ross 708 chicks were evenly divided among three treatments: a basal diet mixed with a synbiotic at 0 (G-C), 0.5 (G-0.5X), and 1.0 (G-1.0X) g/kg. After 15 d, the broilers were exposed to 32 °C for 9 h daily until 42 d. The object memory test was conducted at 15 day; touch, novel object, and isolation tests were conducted at 35 day; tonic immobility (TI) took place at 41 day. At 42 day, plasma corticosterone and tryptophan concentrations and heterophile/lymphocyte (H/L) ratios were measured. Compared to controls, synbiotic-fed broilers, regardless of concentration, had a shorter latency to make the first vocalization, with higher vocalization rates during the isolation test (p = 0.001). the G-1.0 group had the lowest H/L ratio (p = 0.001), but higher plasma tryptophan concentrations and a greater number of birds could reach the observer during the touch test (p = 0.001 and 0.043, respectively). The current results indicate that the synbiotic can be used as a growth promoter to reduce the fear response and stress state of heat-stressed broilers.