Tissue culture is used to multiply Al-Taif rose (Rosa damascena f. trigintipetala (Diek) R. Keller) plants in order to meet the demands of the fragrance, cosmetic, and floriculture industries. The use of arbuscular mycorrhizal fungus (AMF) could potentially improve plant growth and acclimatization performance to ex vitro conditions. Thus, in the current study, we investigated how AMF Rhizophagus fasciculatus influences the growth, establishment, and physiological performance of micropropagated Al-Taif rose plants during the acclimatization stage. The growth and physiological parameters of the AMF-treated plants were evaluated after a 12 week growth period in the growth chambers. The plants treated with AMF exhibited greater height (25.53 cm) and biomass growth values for both shoot fresh weight (0.93 g/plant) and dry weight (0.030 g/plant), more leaves (11.3/plant), more leaf area (66.15 cm2), longer main roots (15.05 cm/plant), total root length (172.16 cm/plant), total root area (64.36 cm2/plant), and biomass from both fresh weight (383 mg/plant) and dry weight (80.00 mg/plant) of the plants. The plants treated with AMF also exhibited increased rates of net CO2 assimilation, stomatal conductance, and transpiration compared to the control plants. The proline content in the leaves and roots was significantly lower in the AMF-treated plants than untreated plants. The Fv/Fm ratio, which serves as an indicator of the intrinsic or maximal efficacy of Photosystem II (PSII) demonstrated a notable decline in the untreated Al-Taif rose plants. These results elucidate the advantageous impact of AMF colonization on micropropagated Al-Taif rose plants, thereby enhancing their resilience against adverse ex vitro conditions.