Background: Malaria is one of the most prevalent public health problems worldwide. Globally, nearly half the world’s population remains at risk of malaria transmitted by the Anopheles mosquito vector. Chemical insecticides used to control the malaria vector are toxic and cause environmental deprivation. Therefore, safe, environmentally friendly, and effective alternative methods are needed to control malaria vectors. The study objective was to isolate and characterize Bacillus thuringiensis (BT) strains and evaluate their insecticidal activity against malaria vectors. Methods: Soil samples were collected from different sites in Amhara Regional State, Ethiopia. Serial dilution was conducted in normal saline; Cutured Coommasie Brilliant Blue staining was performed. BT strains were isolated and characterized based on morphological, microscopic parasporal crystal staining, and biochemical characteristics. Toxicity was assessed by bioassay against Anopheles mosquito larvae. Results/Discussion: Thirteen isolates of Bacillus thuringiensis were identified and characterized based on their morphological, microscopic parasporal crystal staining, and biochemical characteristics. From resuts found that some of the BT strains were 75% effective and while others demonstrate 100% larvicidal potential similar with the reference strain Bti and better than Btk NCIM 2514 within 24 hrs. Conclusions: Five Bacillus thuringiensis isolates showed significantly higher larvicidal efficacy against Anopheles mosquito larvae. Among them, three isolates were promising candidates for future applications in mosquito biocontrol.