Wild medicinal plants are the main source of active ingredients and provide a continuous natural source for many folk medicinal products, a role that is important for society’s health with an impressive record of utilization. Thus, surveying, conserving, and precisely identifying wild medicinal plants is required. The current study aimed to precisely identify fourteen wild-sourced medicinal plants from southwest Saudi Arabia, within the Fifa mountains area located in Jazan province, using the DNA barcoding technique. Two DNA regions (nuclear ITS and chloroplast rbcL) were sequenced and analyzed for the collected species using BLAST-based and phylogeny-based identification methods. Based on our analysis, ten of the fourteen species were successfully identified by DNA barcoding, five were identified as morphologically inspected, and three were morphologically indifferent. The study was able to distinguish some key medicinal species and highlight the importance of combining morphological observation with DNA barcoding to ensure the precise identification of wild plants, especially if they are medicinally relevant and associated with public health and safety usage.