Micropropagation facilitates the rapid production of roses. Nevertheless, in vitro rhizogenesis of essential oil roses has presented significant challenges, primarily attributed to low rates of rooting and poorer acclimatization compared to ornamental rose varieties. This study reports the optimization of in vitro rooting of Al-Taif rose (Rosa damascena f. trigintipetala (Diek) R. Keller) microshoots with the aim of increasing survival rate during acclimatization. We also investigated the effects of various parameters, including type and concentration of auxin (i.e., 2,4-Dichlorophenoxyacetic acid (2,4-D), indole acetic acid (IAA), indole butyric acid (IBA), and naphthaleneacetic acid (NAA) at concentrations of 0, 0.05, 0.1, 0.2, and 0.4 mg/L), salt strength (i.e., full- and half-strength Murashige and Skoog (MS) medium), sucrose concentration (i.e., 20, 30, 40, 60, and 80 g/L), light spectra (a 2:1 or 1:2 blue/red spectral ratio, cool or warm white light at a 1:1 ratio, and fluorescent light), light intensity (photosynthetic photon flux density (PPFD) values of 25, 50, and 100 µmol·m−2·s−1), and activated charcoal (i.e., 0 and 0.5 g/L) on the rooting and growth of in vitro regenerated Al-Taif rose axillary shoots. We found that half-strength MS medium supplemented with 0.2 mg/L NAA, 80 g/L sucrose, 0.5 g/L activated charcoal, and 50 μmol·m−2·s−1 PPFD were the optimal conditions for 100% induction of adventitious roots. Next, micropropagated Al-Taif rose plantlets were successfully transferred to a potting medium containing perlite/peatmoss (in a 1:1 ratio). We found that 98% of plants survived ex vitro conditions. The genetic fidelity of micropropagated Al-Taif rose clones along with their mother plant was tested using the inter-simple sequence repeats (ISSR) molecular marker. The genetic similarity between the micropropagated plantlets and the mother plant of Al-Taif rose plants was 98.8%, revealing high uniformity and true-to-type regenerated plants. These findings may therefore contribute toward the commercial micropropagation of Al-Taif roses.