Since early times, the Tunisian people have used medicinal and aromatic plants as traditional medicine to heal various human diseases. However, few studies have been properly concretized into a document to promote the traditional knowledge of the local population, especially in southern Tunisia. This ethnobotanical survey aims to compile and evaluate the traditional knowledge on different folk use of six medicinal plants: Atriplex halimus (L.), Teucrium polium (L.), Moricandia arvensis L. (DC.), Deverra tortuosa (DC.), Haplophyllum tuberculatum Forssk. (A.Juss.), and Polygonum equisetiforme (Sm). The work is carried out in southeastern Tunisia (Beni Khedache). Ethnobotanical interviews were undertaken during 2021-2022. Forty informants of indigenous communities, aged from 40 to 100 years old, were interviewed. Informants were selected on the basis of their knowledge of medicinal plants and experience in traditional herbal medicine. Interviews were conducted through free listening and semi-structured questionnaires. Collected information were analyzed using frequency of citation (FC), informant consensus factor (ICF), use value (UV), and fidelity level (FL). Findings revealed that Moricandia arvensis L. (DC.) and Deverra tortuosa (DC.) were the two most used species with fidelity level (FL) of 60.00% and 40.00%, respectively. Atriplex halimus (L.), Teucrium polium (L.), and Deverra tortuosa (DC.) achieved the highest scores of frequencies of citation (FC) and use value (UV). Furthermore, all plants were used for the treatment of 15 categories of ailment with predominance of the musculoskeletal (L99) class (ICF, 1.00), and also of the digestive and gastrointestinal system (D99, D70) diseases (ICF, 0.90). The most used plant parts were whole plants (42.08%), followed by leaves (35.00%). Most remedies are prepared by decoction (41.25%), while internal use (orally) (65.42%) was the most frequent administration route. Respondents preferred spring as the most productive harvesting season. These results could set a basis for further pharmacological and phytochemical research and conservative approach of such plant species.