Activation of complex metabolic pathways and antioxidant activities is necessary for enhancing quality and health promoting capacity of food crops. Plant growth regulators are the main factors affecting the expression of different genes and related biological activities. Effect of foliar spray with 24-epibrassinolide (EBL) during growth stages on total antioxidant activity, phenolics, anthocyanins, some anti-stress and antioxidant enzymes activity, fruit quality indices, and pathogen extension in strawberry fruit during cold storage was investigated. EBL significantly enhanced fruit total antioxidant activity, total phenolics, and total anthocyanins contents. Activity of superoxide dismutase, catalase, phenylalanine ammonia-lyase, and polyphenol oxidase enzymes was enhanced by 270%, 81%, 138%, and 94% in response to EBL treatment, respectively. Treated fruits showed lower decay extension, microbial count, weight loss, and higher postharvest life during storage. A positive correlation was observed between the color parameters, total anthocyanins, and antioxidant capacity of the fruits. EBL at 1 µM showed a good potential for enhancing strawberry fruit nutritional and overall quality, phytochemical compounds, and postharvest life.