Objectives: Pseudonocardia alni exhibits antimicrobial activity against tested pathogenic Staphylococcus aureus, Microsporum canis, and Trichophyton mentagrophyte. The present paper aimed to optimize various cultural conditions for antimicrobial metabolite production, purification, and characterization of the active substance. Methods:The effects of various parameters such as culture media, carbon and nitrogen sources, phosphate concentration, pH, temperature, incubation period, and agitation rate on bioactive metabolite production were studied using a flask scale with varying single parameter. The active substances were purified by adsorption chromatography using Silica gel column and Sephadex LH 20 column, and the physical, chemical, and biological properties were characterized. Results:The metabolite production by P. alni was greatly influenced by various cultural conditions. It produced high levels of the antimicrobial substance in International Streptomyces project-2 broth compared with that in potato dextrose broth. The optimum parameters for antimicrobial production from the actinobacterium occurred in the production medium consisting of glucose (1%) and tryptone (1%), 0.001 M of K 2 HPO 4 and 0.05M glycine at initial pH 8.5 and incubated at 30°C for 4 d in stand incubator. The higher concentration of phosphate buffer salts (˃0.01M) repressed the bioactive production. The purified active substance had relative factor R f =0.53 in the mobile phase of a thin layer chromatography system and the maximum absorbance (λ max) at 216 nm. The results of infrared spectra Fourier transform-infrared spectroscopy analysis indicate that it may be regarded to glycopeptide antibiotic. The purified substance had antibacterial and antifungal activities as well as cytotoxic activity in breast cancer cell line Michigan Cancer Foundation-7 and normal hepatic cell line (WRL-68) at a percentage up to 23.7% and 7.64%, respectively. Conclusions:The actinobacterium P. alni was a novel strain having the ability to produce antimicrobial and anticancer substances.