In this study, a total of four endophytic fungi have been isolated from Nigella sativa (Ranunculaceae) seeds growing in Egypt. The isolated fungi were identified morphologically and microscopically up to species to be; Alternaria pluriseptata, Aspergillus tubingensis, Aspergillus parasiticus and Eurotium pseudoglaucus. The extracts of the all identified fungi were screened biologically for antileukemic, cytotoxic, antimalarial, antileishmanial, antimicrobial, antioxidant and anti-inflammatory activities as well as for cannabinoid and opioid receptor binding affinities. All the examined fungal extracts showed good antileukemic activities with Ic₅₀ values ranging from 0.025 to 0.8 µg/ml, while two of them showed cytotoxic activities against Pig kidney epithelial (LLC-PK1) cells. Two fungal extracts exhibited antimalarial activities, three showed antioxidant activities and two exhibited anti-inflammatory activities