Abstarct: Flotation is a mineral separation process where, through preparation, the surfaces of minerals in a pulp are selectively rendered hydrophobic using specific reagents. Gilsonite is a valuable mineral with numerous applications, abundantly found in Iran. Flotation is used to increase the purity of very fine-sized gilsonite. This research employed flotation, along with ultrasonic waves and solutions containing micro-nanobubbles, to improve the separation of gilsonite from impurities at very fine sizes. Therefore, after sample preparation, four series of single-stage (rougher) gilsonite flotation tests and four series of tree analysis tests on the best responses from the rougher tests, with and without the use of ultrasound and micro-nanobubble solutions, were designed using a central composite design. Ultimately, the methods using micro-nanobubbles and ultrasound combined with micro-nanobubbles in the flotation tests resulted in a recovery rate exceeding 90% and a reduction in gilsonite ash content from 54.7% in the initial feed to 16.4% and 18.9% in the concentrate, compared to other methods, respectively.