Chitosan, a linear polysaccharide that can be obtained by partial deacetylation of the natural polymer chitin, possesses exceptional physicochemical properties, including biodegradability and biocompatibility. Apart from the study of chelation with metal ions, there is a small amount of research based on chitosan's connection to other components. The aim of this work is to prepare composite microspheres based on chitosan, Cu2+ ions and bioactive glass using an electrospraying method. The influence of different proportions of copper (II) ions and bioactive glass on the size, shape, morphology and composition of chitosan microspheres was investigated using scanning optical fluorescence and scanning electron microscopy, X-ray analysis and infrared spectroscopy. The surface of the microspheres is wrinkled, non-porous and mostly spherical for all samples except those with the smallest investigated proportion of Cu2+ ions. Upon introducing bioactive glass, noticeable protrusions are observed on the surface of the composite microspheres, which are confirmed by SEM-EDS atomic detection to be bioglass particles. The distribution of particle sizes ranges from 38 μm to 60 μm depending on the composition of each sample. With the increase of Cu2+ ion and bioglass content, the microspheres have a more uniform size distribution and the interval of particle size distribution narrows significantl. FTIR spectra show that the addition of the metal ion leads to a minor difference in the position of the bands corresponding to the O-H and N-H bond vibrations, indicating the interaction between chitosan and copper in these functional groups. Conversely, no significant differences are observed with the addition of bioactive glass. Likewise, the diffraction pattern of chitosan does not change with Cu2+ ions, which confirms the formation of a complex, while a new diffraction maximum characteristic for bioactive glass appears. The morphology and size distribution of composite chitosan-based microspheres obtained by electrospraying method can be controlled with the amount of cupric ions and bioactive glass.