Chitosan is biodegradable, non-toxic and biocompatible material commonly used in tissue engineering and biomedicine. Up to date, few papers have been focused on the investigation of the chitosan–metal ion complex microspheres. Therefore, the aim of this work was to produce chitosan–metal ion microspheres via electrospray method by using chitosans of different deacetylation degrees, and to determine the influence of electric field on the size, shape and microstructure of produced microspheres. Two types of chitosan were used to produce microspheres: chitosan with lower deacetylation degree (MC75) and chitosan with high deacetylation degree (MC95). Copper (ІІ) and zinc (ІІ) ions as divalent metal ions were used to prepare complexes, while ammonia solution served as neutralizing agent. The microspheres where produced at two different voltages: 20 kV and 25 kV. The density (using an aerometer), surface tension (stalagmometer method) and dynamic viscosity (Höppler viscometer) of chitosan and chitosan–metal ion complex solutions were measured. The composition of investigated complexes was determined by X-ray diffraction analysis. The size, shape and size distribution of microspheres were determined using an optical microscope and a light stereomicroscope, while the microstructure was analysed by a scanning electron microscope. Biological characterization of microspheres was performed by cytotoxicity assay on human embryonic cells during three days of culture. The surface tension and dynamic viscosity results indicated easier electrospraying production of regular microspheres from MC75–metal ion complexes. The identification of chitosan-ion complexes indicated interactions between Cu2+ and Zn2+ ions and chitosan, respectively, without the formation of inorganic crystalline phases. The microscopic analyses of produced microspheres showed a narrow size distribution for microspheres produced at 25 kV. Additionaly, the more spherically shaped microspheres were detected for complexes with chitosan MC75 with respect to the complexes obtained from MC95. Scanning electron microscopy showed highly porous structure of microspheres produced at both voltages. The cytotoxicity test showed no toxicity of microspheres of MC75–Zn2+ complexes, while microspheres MC75‒Cu2+ higher concentrations were cytotoxic for human embryonic kidney cells Hek293.