Objective To isolate, identify and evaluate the antioxidant, antimicrobial, hepatoprotective potentials, total phenolic content, flavonoid content, tannin content of ethyl acetate extract of endophytic fungus Achaetomium sp., isolated from Euphorbia hirta. Methods Hepatoprotectivity of ethyl acetate extract of Achaetomium sp., was evaluated by CCl4 induced toxicity in HepG2 cells and subsequently analysed for cell viability using MTT assay. It also demonstrates antioxidant and antimicrobial potentials by DPPH radical scavenging assay and well diffusion assay respectively. Quantification of total phenolic content, tannin content and flavonoid content were assessed by spectroscopic methods. Results Phenols, flavonoids and tannins were the phytochemicals present in ethyl acetate extract of Achaetomium sp., with rich phenolic content exhibited potent hepatoprotective, antimicrobial and antioxidant activities. The hepatoprotective activity was recorded as of 72.13% ± 2.948% of cell viability at a concentration of 150 μg/mL, whereas the standard silymarin showed 93.260% ± 0.784%. It was observed to be dose dependent, when CCl4 exposed HepG2 cells were treated with different concentrations of ethyl acetate extract. Antibacterial activity showed significant inhibition against Staphylococcus aureus, Pseudomonas aeroginosa, and Klebsiella pneumoniae. The antioxidant activity ranged from 66.890% ± 1.385% to 87.340% ± 0.289% with (44.02 ± 1.57) μg of total phenolics, (54.54 ± 1.82) μg of flavonoid content and (18.790 ± 1.018) μg of tannin content. Ascorbic acid, BHT (butylated hydroxyl toluene) Gallic acid and Pyrogallol were used as standards which showed 98.370% ± 0.763%; 97.080% ± 0.636%; 94.890% ± 1.103% and 96.980% ± 0.098% reducing potential respectively. Conclusions The results reveal that the metabolites produced by endophytic fungi isolated from Euphorbia hirta could be novel natural products that could lead to new drug discovery. © 2017 Hainan Medical University