The present study aimed to assess plant diversity and vegetation structure of Gebel Maghara at different habitats. Floristic changes with emphasis on medicinal plants and human impacts are also considered. Twenty five sites representing different localities and habitats of Gebel Maghara were surveyed for vegetation measurements, soil sampling, and human activities description. Different diversity indices were measured, and multivariate analyses were carried out using TWINSPAN for classification and CANOCO for ordination. Soil physical and chemical properties were analyzed in 40 surface soil samples. The results revealed that Maghara area is characterized by high and unique plant diversity. One hundred and ten species are recorded in Maghara area including 70 medicinal species. W. Maghara and W. Aroseya are richer in medicinal plants than other localities. Seven main vegetation groups are recognized in the area. The dominant species in these groups are Anabasis articulata, Juniperus phoenicea, Zilla spinosa, Zygophyllum dumosum, Retama raetam, Artemisia monosperma, and Acacia pachyceras var. najdensis. The vegetation group dominated by Juniperus phoenicea is more diverse than the others. Sand and clay fractions, total carbonate and pH are the main soil conditions controlling the distribution of plant communities in the area. Gorges and slopes are the most diverse habitats. Beta diversity between previous (1960) and current (2005) surveys reflects a large floristic change. About 121 species previously recorded since 1960 are not recognized in the current survey and many of them may be lost. In addition to aridity conditions, human impacts including mining and quarrying, over collection of vegetation for different purposes, and cultivation are considered the main threats of plant diversity in the area. The study recommends that action plans to protect the Maghara's plant diversity should be based on improved knowledge of the biodiversity, empowering local people to become more involved in decision making, improvements in local and national governance, and more effective operational mechanisms for implementing protection of plant diversity at identified hotspots rich in medicinal and/or endangered species.