Aims: The aim of this study was to isolate the endophytic fungi from Catharanthus roseus and screen the cytotoxicity and antimicrobial activity of alkaloids extracted from these fungi. Methodology and results: A total of 56 endophytic fungal isolates were screened from C. roseus plant parts. Aspergillus fumigatus and Fusarium oxysporum were the most frequent species. Determinations of alkaloids extracted from the most dominant endophytic fungal species were done. The highest significant total alkaloids productions were by A. fumigatus and Aspergillus niger, while the least significant one was by Botrytis cinera. Antimicrobial assay of endophytic fungal extracts indicated that both A. niger and F. oxysporum exhibited significant antimicrobial activities, while A. fumigatus exerted the least activity. In vitro cytotoxicity assay of the endophytic fungal extracts was done against human breast cancer (MCF-7) and liver cancer (HEPG-2) cell lines using SRB assay method. A. niger extract showed potential cytotoxic effect on MCF-7 cell line with IC50 value of 42.1 μg/mL, while the least cytotoxic effect was exhibited by F. oxysporum on MCF-7 cell line with IC50 value of 66.9 μg/mL. Gas Chromatography Mass Spectroscopy (GC-MS) was used for analysis of alkaloids in mycelial and filtrate extracts of A. niger, where eleven compounds were detected. Conclusion, significance and impact of study Alkaloids extracted from Catharanthus roseus associated endophytic fungi had cytotoxic and antimicrobial activities.