Gummy Stem Blight (GSB), caused by Stagonosporopsis sp., is a severe disease affecting cucurbits. It impacts all above-ground parts of the plant at any growth stage, leading to significant yield losses worldwide. Managing GSB is challenging due to the limited availability of resistant cultivars. Conventional strategies rely heavily on fungicides; however, their effectiveness has declined due to the pathogen’s ability to develop resistance to site-specific fungicides. This study aims to characterise the fungal pathogens associated with GSB-infected plants and assess the efficacy of fungicides used in industrial applications. A large collection of fungal isolates were cultured from diseased cucumber plants from New South Wales and South Australia and identified as GSB using Sanger sequencing and bioinformatics tools. These isolates were then tested for fungicide resistance against various products used by industry at concentrations based on manufacturer recommendations, along with diluted versions of these concentrations. The fungicides were tested at four different concentrations to evaluate their effectiveness. The results revealed varying levels of resistance among GSB isolates. Some isolates exhibited complete resistance to the full (100%) concentration of fungicides, while others were susceptible at this concentration but not at the lower, diluted doses. Notably, one species demonstrated complete resistance to major fungicides, highlighting the urgent need for optimised disease management strategies. This study contributes to the development of more effective control measures by tapping into the genetic variability found within fungal culture collections and emphasises the importance of monitoring fungicide concentrations. This research enhances our understanding of fungicide resistance in GSB, providing a promising strategy for better managing diseases in cucurbit crops.