Nowadays, the need for new and useful pharmaceutical compounds, especially those with natural source and without side effects are felt more than ever for treatment of various human diseases. Development of resistance to antibiotics in bacteria, the emergence of life-threatening viral diseases and growing fungal infections in human societies, reflects the inefficiency of current methods to deal with these medical problems. Fungi have provided a potential opportunity to obtain antimicrobial compounds. The fungal kingdom provides abundant and diverse source of bioactive metabolites for developing the drugs and agrochemical pesticides. In order to isolate novel bioactive compounds, during a survey in May 2017, samples were collected from walnut trees with typical Cytospora canker symptoms, in West Azerbaijan province, Iran. Isolation and purification of the causal agent was carried out using routine plant pathology techniques. The identity of the Cytospora isolates were confirmed based on morphological features and sequence data of ITS-rDNA regain and TEF gene. In order to prepare crude extract, selected isolate was fermented in Q6 and rice media. After separation of mycelia, supernatant was treated with XAD-16 resin. Then, XAD and mycelia were extracted with acetone and EtOAc. In the case of rice medium, it was extracted using EtOAc. Fractionation and purification was performed by preparative HPLC using ACN+0.05% TFA (solvent B) and H2O+0.05% TFA (solvent A). Antimicrobial activities and cytotoxicity assays of the isolated metabolites were performed on 12 pathogens and mouse fibroblast cell line L929 and mammalian HeLa KB3.1 cancer cells, respectively. Six (6) new metabolites including four terphenylquinones and two dicarboxylic acid derivatives were isolated and elucidated along with five known compounds by NMR spectroscopy and mass spectrometry. The isolated compounds showed range of antimicrobial activity against the tested fungi and bacteria. Significant cytotoxicity was observed for terphenylquinone derivatives against mouse fibroblast L929 and mammalian HeLa KB3.1 cell lines with IC50 values of 2.4 to 26 µg/ml.