Aim: Due to the urgent need for anticancer agents, investigation of endophytes of medicinal plants growing in special environments is considered a promising approach for the search of bioactive natural products. Materials and Methods: The ethyl acetate extracts of the cultures of twelve endophytic fungi were isolated from the medicinal plants Calotropis procera fam Apocynaceae, Solanum nigrum fam Solanaceae and Forsskaolea tenacissima fam Urticaceae growing in one of the richest areas of plant biodiversity in Saudi Arabia, Najran. All isolated and identified endophytic fungi were subjected to preliminary screening assays for cytotoxic activity. Results: Four of the isolated endophytic fungi (Cladosporium herbarum (1), Hortaea werneckii (2), Penicillium solitum (3) and Eurotium chevalieri (4)) exerted cytotoxic activity against HepG2, T-47D, HCT-116 and RAW 264.7 in the MTT cell viability assay. The highest cytotoxic effects were observed for the extract of Penicillium solitum on HepG2 and HCT-116 cell lines with IC50 values of 13 μg/ml and 42 μg/ml, respectively. All endophytic extracts showed rather weak cytotoxic effects against RAW 264.7 cell line with IC50 values of 357, 347, 345 and 420 μg/ml for extracts of endophytes 1,2,3 and 4, respectively. High cytotoxic effects were observed for the extracts of endophytes 1, 2 and 3 against T-47D cell line with IC50 values of 78, 69 and 94 μg/ml, respectively. Conclusion: These bioactive endophytes represent candidates for future investigation of bioactive metabolites with potential medical applications.