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Ascosphaera apis (Onygenales: Ascosphaeraceae) is a chalkbrood causing pathogen in honeybees. Recently, chemicals have been used against this disease. Consequently, the abuse of chemicals has caused serious impacts on the environment and honey quality and on honeybee colonies themselves. Therefore, the search for biological control options should be the priority for controlling chalkbrood disease. In this experiment, we employed restriction enzyme mediated integration (REMI) to construct mutants (transformants) with growth inhibition on A. apis in honeybees using the Trichoderma atroviride T23 wild strain. The integration of the plasmid pBluescript KS-EH1 into the REMI mutants was confirmed by PCR and Southern hybridization. As a result, ten single-copied T. atroviride T23 transformants were successfully identified. Furthermore, culture filtrates of the transformants, their wild strain and Shabaijing (a commercial fungicide, ethyl ethylsulfonothiolate) were tested to compare their in vitro growth inhibition capability on A. apis . The results demonstrated that transformants Tat5 and Tat6 had the highest growth inhibition activity with the minimal fungicidal concentration (v/v) of 3.5–4.5%. Thus, the Trichoderma transformants constructed by REMI could be used as potential biological agents against the growth of fungal diseases in bee hives. Further refinement and strategy development for the integrative use of this result are vital future plans. [ABSTRACT FROM AUTHOR]