Fusarium wilt, incited by the fungus Fusarium oxysporum f. sp. niveum, is a soilborne disease that affects watermelon production worldwide. Approaches for effective management of Fusarium wilt in watermelon are limited. Studies conducted in recent years indicated that prothioconazole and thiophanate-methyl reduced the disease significantly under field conditions. However, effects of the fungicides on different life stages of F. oxysporum f. sp. niveum and potential existence of fungicide resistance in F. oxysporum f. sp. niveum populations are unknown. In the present study, effects of prothioconazole and thiophanate-methyl on mycelium growth and spore germination of F. oxysporum f. sp. niveum isolates collected in watermelon fields in Georgia were determined. In vitro mycelium growth studies indicated that all 100 isolates evaluated were sensitive to prothioconazole; the effective concentration that suppressed mycelium growth by 50% ranged from 0.75 to 5.69 μg/ml (averaged 1.62 μg/ml). In contrast, 33 and 4% of the isolates were resistant to thiophanate-methyl at 10 and 100 μg/ml, respectively. Microconidial germination assays showed that 36 and 64% of the isolates tested were sensitive or intermediately sensitive to prothioconazole at 100 μg/ml but the fungicide did not inhibit spore germination at 10 μg/ml. Sequencing a portion of the β-tubulin gene of eight isolates resistant or sensitive to thiophanate-methyl indicated that fungicide resistance was associated with a point mutation at nucleotide position 200, resulting in a substitution of phenylalanine by tyrosine. This is the first report of isolates of F. oxysporum resistant to thiophanate-methyl. Results of the research suggest that prothioconazole may be a viable option for management of Fusarium wilt of watermelon whereas thiophanate-methyl should be used judiciously due to the existence of isolates resistant to the fungicide.