Selecting the right rootstock is crucial for successful fruit production and managing both biotic and abiotic stresses in commercial fruit orchards. To enhance the resilience of peach orchards, this study evaluated the physiological and biochemical responses of Prunus spp. rootstocks , ‘Flordaguard’ and ‘MP‐29,’ under normoxia (sufficient oxygen content) or short‐term hypoxia (low‐oxygen content) and ambient or high temperature (40°C) in the root zone. Physiological responses measured were net photosynthesis, stomatal conductance, transpiration, intercellular CO2 concentration, water use efficiency, the leaf chlorophyll index, and the maximum potential quantum efficiency of photosystem II. The leaf concentrations of nitrogen, phosphorus, potassium, magnesium, calcium, sulfur, boron, zinc, manganese, iron, and copper were also analyzed. Reactive oxygen species (ROS) and antioxidants analyzed were superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, ascorbate peroxidase (APX) activity, glutathione peroxidase (GPX) activity, proline content, glycine betaine content (GB), lipid peroxidation (LPO), superoxide (O2−) concentration, and hydrogen peroxide (H2O2) concentration. When subjected to root zone hypoxia or high temperature individually, ‘MP‐29’ performed better physiologically than ‘Flordaguard’. However, when root zone hypoxia and high temperature were combined, ‘MP‐29’ performed better biochemically with enhanced antioxidant activity, osmolyte content, and nutrient absorption. Nutrient analysis of leaves revealed that ‘MP‐29’ had higher N, P, K, Ca, and B concentrations than ‘Flordaguard’. Consequently, ‘MP‐29’ demonstrated greater tolerance to short‐term exposure to the combined effects of high root zone temperature and hypoxia. This research contributes to identifying a suitable rootstock within the Prunus genus able to withstand root zone conditions that often result from severe weather events commonly experienced in Florida and other parts of the world.