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The characteristics and stability of ozone nanobubbles (NBs) were investigated for the first time under different preparation conditions and freshwater conditions (i.e., pH, natural organic matter [NOM], carbonate, calcium, and temperature) for an extended period. Two oxygen gas flow rates (4 and 1 L/min) used in ozone NB generation affected the characteristics and stability of ozone NBs. The ozone NBs generated at a high initial dissolved ozone (12.5 mg/L) concentration showed a much higher brightness during measurements than the ozone NBs generated at a low initial dissolved ozone concentration (1 mg/L). The former also exhibited a higher negative surface charge and higher stability in comparison to the latter. The stability and half-lives of ozone NBs followed the order of 3 mM Ca 2+ < pH 3 < NOM with high specific ultraviolet absorbance at 254 nm (SUVA 254 = 4.1 L/mg·m) < pH 7 < pH 9, while the effects of carbonate and temperature were insignificant. Ozone NBs were relatively stable in waters for a long period (e.g., ≥ 60 days) except for high hardness or low pH conditions. Higher levels of hydroxyl radicals were produced from ozone NB solutions as compared to conventional ozonation.