Recent studies of bioactive agents, simultaneous encapsulation in chitosan/alginate microcapsules revealed that encapsulation in the same compartment does not inhibit activity either of Trichoderma viride spores nor copper cations. The objective of this work was to investigate the influence of formulation variables (concentration of copper cations, chitosan layer and microcapsule size) on T. viride spores release. Results showed that the increase in copper cation concentration promoted, but the increase in microcapsule size and presence of the chitosan layer on microcapsule surface reduced T. viride spores release. Fitting to simple Korsmeyer–Peppas empirical model elucidated the underlying mechanism of release. Fickian diffusion controlled release from microcapsules without chitosan layer and smaller microcapsules with chitosan layer, whereas anomalous diffusion mechanism (a combination of the diffusion and erosion mechanisms) was found to be the rate-controlling mechanism from larger microcapsules with chitosan layer. The investigation showed that proper selection of formulation variables helps in designing microcapsules with the desirable release of T. viride for plant protection and nutrition.