High temperatures significantly impact grapevine growth and development and lead to severe losses in grape quality and production. To minimize the impact of those environmental constraints, the application of biostimulants (BSts) has emerged as one of the most interesting strategies. BSts application derived from cactus species has been described as a successful approach to enhance tolerance to biotic and abiotic stresses. In this study, an aqueous extract prepared from the cladodes of Opuntia ficus-indica was applied through foliar spraying to grapevine plants (Vitis vinifera L.) ’Aragonez’ already under heat stress. The effect of the extract application on protecting grapevine plants against heat stress was assessed in an experiment running during 15 days after extract application by determining several physiological parameters and detecting the changes in the whole proteome profile by comparing non-treated and extract-treated samples. Results show that physiological parameters directly related to photosynthesis showed a positive effect of the extract in mitigating heat stress in grapevines. Proteomic analysis indicated that the extract significantly upregulated proteins associated with photosynthesis and stress responses. This study provides new insights about the effect of O. ficus-indica extract in grapevines, offering a valuable strategy for future applications under field conditions.