Non-military threats can be in the form of disease outbreaks, biological attacks, or forms of threats that endanger state sovereignty, territorial integrity, and national safety. Threats that come from disease outbreaks carried by animals can become biological weapons. The spread of Wolbachia mosquitoes for the first time in Bantul and Sleman Regencies of Yogyakarta Province was one of the efforts to reduce the number of dengue fever, but the spread of Wolbachia mosquitoes was rejected by the community. The purpose of this study was to analyze the current spread of Wolbachia mosquitoes, the impacts caused, challenges in the spread of Wolbachia mosquitoes, and the implications of the spread of Wolbachia mosquitoes in Bantul and Sleman Regencies for Indonesia's non-military defense. This research used a qualitative approach with a case study method. The results of this study are the spread of Wolbachia mosquitoes improves public health in Bantul and Sleman Regencies, the impact of the spread of Wolbachia mosquitoes in the short term can reduce the rate of dengue fever by 77% in Bantul and Sleman Regencies, the challenge in the future is to provide education and socialization to the community at large, and the implications for Indonesian non-militery defense are the need for cross-sector collaboration and the role of the Indonesian Army to mitigate in the context of early prevention. The suggestion of this research is to encourage the strengthening of biotechnology defense in the framework of Indonesia's nonmilitary defense.