Micropropagation is currently one of the primary methods for plant propagation, known for its efficiency in producing disease-free and vigorous plants. However, the final stage of this—acclimatization, is critical due to the transfer from a controlled in vitro environment to an external one. To reduce mortality and alleviate acclimation stress, plant growth regulators (PGRs) or biostimulants can be employed. This study investigated the effects of exogenously sprayed PGRs: 0.001 mg L−1 abscisic acid (ABA), 0.001 mL L−1 brassinolide (BL), 0.001 mL L−1 24-epibrassinolide (24-epiBL), and 0.3 mL L−1 biostimulant Goteo on the physiological and biochemical responses of Echinacea purpurea ‘Raspberry Truffle’ plantlets during the acclimation process. The effects of treatments at various acclimation stages on chlorophyll (chl) and carotenoids content, hydrogen peroxide (H2O2), catalase (CAT), malondialdehyde (MDA), free amino acids, soluble proteins, total soluble sugar and reduced soluble sugars were tested in this research. The results confirmed changes in biochemical parameters, including an increase in chlorophyll and carotenoids in the acclimatization period where the highest level obtained by BL spraying. A decline in stomatal conductance was also observed, where ABA influenced the most on drop. It was also recorded the decrease in H2O2 and MDA concentration. CAT activity increased, especially with biostimulant treatment. We recorded an increase in total soluble proteins along acclimatization. Goteo affected the most on morphology parameters, ABA, BL and 24-epiBL increased acclimatization efficiency. Our studies indicate that potentially the most effective substances in the acclimatization of E. purpurea ‘Raspberry Truffle’ are brassinosteroids and ABA.