Chlorine dioxide (ClO2) is a disinfestant used to control pathogens in water. To determine if interactions between inorganic ions and pH levels effect ClO2 activity in vitro, concentrations of ClO2 (0, 1, 3, 5, 7, 9, 22, 24, 46, 58, and 70 mg/liter) were mixed for 10 min in solutions containing a nitrogen and hard water solution with equal concentrations of ammonium, nitrate, and synthetic hard water (0 and 100 mg/liter) and a divalent metal ion solution with equal concentrations of copper, iron, manganese, and zinc (0, 1, 3, and 5 mg/liter) at pH 5 and 8. Macro- and microconidia of Fusarium oxysporum f. sp. narcissi or conidia and aleuriospores of Thielaviopsis basicola were injected into each suspension for 30 s, captured on filter paper disks that were flushed with water, and plated on 50% potato dextrose agar. Spore germination was quantified after 1 day. ClO2 activity had a similar effect on both fungal species and all types of propagules with interactions among the divalent metal ion solution, nitrogen and hard water solution, and pH treatments. A higher concentration of ClO2 was required at pH 8 than at pH 5 to achieve a lethal dose resulting in 50% mortality of spores (LD50). The addition of the divalent metal ion solution required an increase in ClO2 concentration to maintain a LD50. When combined with the nitrogen and hard water solution, the divalent metal ion solution placed a higher demand on ClO2 at pH 5 and a lower demand on ClO2 at pH 8, thus requiring an increase and decrease in a ClO2 concentration, respectively, to achieve a LD50. Chlorine dioxide doses resulting in 50% mortality ranged from 0.5 to 7.0 mg/liter for conidia of F. oxysporum, 0.5 to 11.9 mg/liter for conidia of T. basicola, and 15.0 to 45.5 mg/liter for aleuriospores of T. basicola.