Serapias vomeracea is an economically important orchid species which is over-collected from nature, because of its glucomannan-rich tubers. Thus, optimization of in vitro culture methodology in this species is required to meet industrial needs and to secure its populations in nature. This study aimed to optimize the surface sterilization protocol for S. vomeracea seeds and to select the optimal seed germination medium by comparing the commonly used media in in vitro orchid culture. During seed surface sterilization, ethyl alcohol (EtOH) pre-treatment prior to sodium hypochlorite (NaOCl) treatment increased the disinfection success and viable seed yield when examined using the triphenyl tetrazolium chloride (TTC) seed viability test. Also, low-g force centrifugation as an additional step in the surface sterilization method separated the seeds without embryo from the viable seeds and thereby decreased potential counting errors after incubation. Comparison of media showed that solid Knudson C (KN) medium induced the highest number of germinated seeds. However, seed germination success of Lindemann (LN) and Vacin & Went (VW) media was found to be higher when the media was used in liquid form. Half-strength liquid VW was the only medium that induced higher germination success than the other full-strength media. The highest number of ungerminated seeds was found when using KN medium whereas liquid VW medium gave the lowest number. In general, protocorm formation was triggered when the media were used in liquid form. However, rhizoid elongation was suppressed in liquid media. These findings suggest that this optimized seed surface sterilization method offers a simple and effective alternative to classical methods. Additionally, solid KN medium may be considered as a cost-effective and reliable alternative to other commonly-used complex media in S. vomeracea cultures.