BtPlus is a group of biopesticides that are made of Bacillus thuringiensis and immunosuppressant. A new BtPlus that exhibits high insecticidal activity against mosquito larvae has been investigated in control efficacy in field conditions and its environmental safety against aquatic system. This study assessed the control efficacy of BtPlus against mosquito larvae with two different application methods. In aerial spraying application (100 mL per 3.3 m 2 ), BtPlus was effective at 50% or above formulation concentrations to control mosquito larvae. For a direct application to aqueous mosquito habitat, a semi-field mimicking paddy rice field was constructed. In this condition, BtPlus showed 80% and 100% control efficacies at 0.1% and 0.2% concentrations, respectively. BtPlus also showed 40% mortality against adults at 0.1% concentration in 10% sugar bait. However, its control efficacies against adults were much less than against larvae. Safety assessment of BtPlus against ecosystem was evaluated using young carp (Cyprinus carpio), a water flea (Daphnia magna), and a honey bee (Apis mellifera). BtPlus did not give any adverse effects on these nontarget organisms. Based on these results, BtPlus can be applied to control mosquitoes by direct aqueous application to paddy rice field.