This work was carried out during two successive seasons (2016 and 2017) on cucumber fruits from a plastic greenhouse and from open field cultivation in El Gharbeia and El Giza Governorates, Egypt. Isolation trials from spoilage fruit samples of plastic greenhouse cultivation recorded high frequency of Alternaria tenusinium, Fusarium spp. and Pleospora alli. The most common fungi of rotten cucumber fruits from an open field were Galactomyces spp. and Fusarium spp. Pathogenicity tests proved that, Fusarium solani from El-Gharbeia followed by A. tenusinium from El-Giza were the most frequent isolates responsible for rot of cucumber fruits from plastic greenhouse cultivation. Moreover, the most frequent isolates causing postharvest disease of cucumber fruits of the open field were Galactomyces candidium from El-Giza followed by Geotrichum sp. and F. fujikuroi from El-Gharbeia Governorates, respectively. This is the first report of several fungi causing postharvest fruit rot disease of cucumber i.e., G. candidium, Geotrichum sp., A. tenusinium, P. alli and Fusarium spp. (F. fujikuroi, F. verticiolides, F. solani, F. geraminearium and Fusarium incarnatum). Fungal isolates were identified according to cultural, morphological and molecular characterization based on sequencing of internal transcribed spacer1 (ITS1). All the ITS nucleotide sequences of fungi were applied and conserved in GenBank.