Halocnemum strobilaceum halophyte occupies tidal and coastal environments in Egypt. Endophytes have the potential to yield novel and important natural products. Herein we evaluate the effect of changing media on the production of secondary metabolites by Halocnemum strobilaceum endophytes. Endophytic fungal ethyl acetate extracts of different culturing media; corn “Zea mays" (CGA), rice “Oryza sativa" media (M1), cowpea “Vigna unguiculata" (M2), painted pony “Phaseolus vulgaris" (M3) and broad bean “Vicia faba"(M4), were screened to evaluate their cytotoxic and antimicrobial activities. Media showing the highest antibacterial activities of fungal metabolites were riceculture media with Penicillium citrinum E-346 and broad bean media with Aspergillus flavus RF-03with MIC (Minimum inhibitory concentration) value of 0.94 µg/mL for both against Bacillus subtilis (ATCC-6633). No antifungal activities against Candida albicans or Aspergillus niger were noticed with any of the tested fungal strains. The cytotoxicity assays using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method against the breast cancer cell line (MCF-7) manifested a good safety index with all media. This evaluation aims to select the best growth conditions for the isolated endophytic fungi to direct further studies toward the best media for chemical investigation of their antimicrobial metabolites.