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The strawberry fruit (Fragaria x ananassa) is a thin-skinned fruit that is easy to spoil due to mold, dehydration, and structural loss because of continuous metabolism processes even after harvesting. This study focused on isolating and identifying fungal pathogens of hydroponic strawberry fruit and investigating the antifungal effect of chitosan solution (CTS), and CTS in combination with calcium gluconate (CaGlu) and polyvinyl alcohol (PVA) towards the identified pathogens). Six strains of pathogens were isolated and identified were Aspergillus fumigatus, Aspergillus niger, Botrytis cinerea, Colletotrichum gloeosporioides, Penicillum chermesinum, and Rhizopus stolonifer. In addition, the results have shown a high antifungal effect of CTS 1% (w/v) to the 6 fungal strains with efficiency values between 53.54% and 77%. Higher effects were observed in the case of CTS 1% combined with CaGlu 0.5% (w/v) as efficiency the was increased from 73.96 to 93.33% and highest effect was obtained with the solution containing all the 3 agents: CTS 1%, CaGlu 0.5%, and PVA 0.39% (w/v) (up to 79.38 - 96.88% efficiency). This result provides an important scientific basis for the development of preserving membrane for hydroponic strawberry in order to improve the fruit quality after harvesting and during storage and transportation.