This study describes 32 fungal endophytes isolated from different tissues of Brugmansia aurea Lagerh. Each fungal strain was authenticated based on internal transcribed spacer rDNA sequence. Phylogenetic analysis showed that these fungi are distributed in three classes, seven orders and 12 genera. The dichloromethane extracts of endophytic strains were screened for anticancer and antimicrobial activity. Anticancer activity of extracts against human cancer cell lines revealed that 50% strains are active with IC50 < 10 μg/mL. While analysing antimicrobial potential against both Gram-positive and Gram-negative bacteria, 56.25% endophytic strains displayed activity at least against one of the tested human pathogenic bacteria with minimum inhibitory concentration of 12.5-100 μg/mL. In vitro antagonistic activity of endophytes was analysed against Sclerotinia sp., Aspergillus fumigatus, Fusarium solani, A. flavus and F. oxysporum pathogen. The broad-spectrum anti-phytopathogenic activity was shown by R2BA. The presence of ketoacyl synthase domain of polyketide synthase gene and high degree of bioactivity shown by endophytic fungi suggested that they have potential to produce therapeutic compounds and to serve as biocontrol agent.