The aim of the study was to develop an efficient micropropagation protocol ofOriganum scabrum, which will allow its commercial exploitation in the pharmaceutical and horticultural industries. First, the effect of the date of the explant collection (20 April, 20 May, 20 June, 20 July, 20 August) and the position of the explant on the plant stem (shoot apex, 1st node, 3rd node, 5th node) on the establishment of in vitro cultures was studied (1st experiment: Stage I). Next, the effect of temperature (15 °C, 25 °C) and the node position (microshoot apex, 1st node, 5th node) on the microplant production and ex vitro survival of plantlets was studied (2nd experiment: Stage II). The optimum season to collect explants from wild plants was shown to be during the vegetative growth of the plants (April to May), while the shoot apex and the 1st node were the most suitable explants. For the proliferation and production of rooted microplants, the best results were obtained from single-node explants excised from microshoots produced from 1st node-explants collected on 20th of May. Temperature did not affect microshoot number, leaf number and the percentage of rooted microplants, while microshoot length was higher at 25 °C. Moreover, microshoot length and the percentage of rooted microplants were higher in those derived from apex explants, while the survival of plantlets was not affected by treatments and ranged between 67% and 100%.