The Opuntia genus is widely recognized as a significant member of the Cactaceae family. The eastern Moroccan prickly pear’s wild ecotype is renowned for its production of fruits of superior quality, which are in high demand. Nonetheless, the white cochineal (Dactylopius opuntiae) has emerged as a significant hazard to the persistence of the indigenous wild prickly pear population in the majority of the country’s territories. Conventional plant propagation techniques may facilitate the transmission of pathogens to successive generations and thus fail to satisfy market requirements. Therefore, the primary goal of this study was to develop a rapid and efficient protocol for large-scale production of the eastern Moroccan wild ecotype using axillary buds as the starting material. Sterilization of the starting material is a crucial but challenging step in this species, as the meristem is located just beneath the spine. The protocol developed for this study produced moderately satisfactory results, with 20 to 30% contamination in each experiment. The obtained shoots were incubated on Murashige and Skoog medium supplemented with varying concentrations of BAP (0, 1.5, 3, 5, and 7.5 mg/L). The treatment with BAP at 5 mg/L exhibited a statistically significant increase in the average number of regenerated shoots per explant (19.42). The effect of kinetin on the rooting of prickly pear was evaluated by transferring the shoots to a MS medium supplemented with varying concentrations of kinetin (0, 0.5, 1, 1.5, 2, and 2.5 mg/L). The use of kinetin increased the number and length of roots while also shortening the root development period from 21 days to 10 days. The best results were obtained at a concentration of 1.5 mg/L of kinetin. Furthermore, satisfactory acclimatization of plants was achieved by using plastic containers with a gradually increasing opening of the lids. The outcomes of this experimentation have significant potential to facilitate the preservation of this botanical variety, reduce the risk of white cochineal infestation, and address the need for superior quality fruit supply in the market.