A study was conducted to assess the efficacy of prebiotic and herbal supplements to relieve transportation stress based on changes in physiological, hematological, and molecular responses in Kenguri sheep. Thirty healthy female sheep were randomly divided into three groups: a control group (CKS) with no supplementation, a prebiotic supplementation group (PKS), and an herbal supplementation group (HKS). The animals were transported 230 km over seven hours during summer conditions, with temperatures ranging from 32.5 °C to 34.9 °C. The groups that received the prebiotic (75.6 breaths/min; 64.8 beats/min) and herbal supplementation (31.0 breaths/min; 66.8 beats/min) had a significantly reduced respiration rate (RR) and pulse rate (PR) compared to those of the control group (38.7 breaths/min; 75.6 beats/min) ( < 0.01 and < 0.05, respectively), indicating improved physiological stability. The hemoglobin (HGB) and hematocrit (HCT) levels were also significantly lower in the PKS (24.2 g/dL; 24.8%) and HKS (24.7 g/dL; 24.5%) groups than in the CKS (28.1 g/dL; 24.9%) ( < 0.05), highlighting the mitigation of hematological stress. Further, the plasma glucose level was significantly higher ( < 0.01) in the HKS group (80.0 mg/dL) compared to the CKS group (63.5 mg/dL). However, rectal temperature (RT) and skin temperature (ST), red blood cells (RBCs), mean corpuscular volume (MCV), and white blood cells (WBCs) showed no significant differences among the groups. These findings demonstrate that prebiotic and herbal supplementation can effectively reduce transportation-induced stress in Kenguri sheep, offering a practical strategy to improve the welfare and resilience of livestock under challenging environmental conditions.