The main aim of our study is to investigate the anticancer potential of our cultivated entophytic fungal strains from Nigella sativa seeds. The strains were identified by sequencing of the partial 18S rRNA gene and the internal transcribed spacer (ITS) region as Aspergillus sp. (SA4), Penicillium sp. (SA5), and Fusarium sp. (SA6). We carried out metabolic profiling for three fungal strains to investigate their metabolites diversity.Profiling of the different extracts revealed their richness in diverse metabolites and consequently fourteen compounds (1-14) were annotated. In addition, the obtained extracts were examined against three cell lines HepG2, MCF-7 and Caco-2 showed activity with IC50 values in the range of 1.95-39.7 μg/mL. Finally, molecular docking study was performed showing questinol as the lowest glide binding score value (-5.925 kcal/mol) among all identified compounds. Our results showed Nigella sativa-associated endophytes as a promising source for further studies to look for anticancer secondary metabolites.