This investigation aimed to identify the fungal pathogen responsible for gray mold in immature cucumber fruits and evaluate the influences of fennel essential oil (FEO) and nano-emulsion (FEO-NE) on Botrytis cinerea Pers: Fr., the causal agent of gray mold. Botrytis cinerea was identified as the fungal isolate through molecular characterizations obtained from sequencing of Internal Transcribed Space (ITS). The nucleotide sequence of fungi was registered in Gene Bank under accession number PP758474. Ultra-sonication based nano-emulsions containing fennel essential oil can act as antifungal agents, specifically for the production of plant-derived emulsions. Therefore, the aims of this study were to evaluate the efficacy of fennel nano-emulsion (FEO-NE) and essential oil (FEO) on the mycelial linear growth of B. cinerea at concentrations of 500, 250, 125, 65, and 30 µg ml-1, compared to essential oil. The FEO-NE was the most effective in reducing the mycelial linear growth of B. cinerea isolate at a concentration of 30 µg ml-1. The reduction rate in mycelial linear growth of fungi using FEO-NE was 13.87% and at the same concentration, FEO was 1.85% compared to the control treatment. In vitro the results were consistent with the in vivo results, showing that FEO-NE was better than FEO in terms of disease severity on fruits, with a ratio of 0.55 for FEO-NE and 0.83 for FEO compared to the control treatment. The essential oil of fennel was identified using Gas Chromatography-Mass Spectrometry (GC–MS); the main compounds identified were Fenchone (8.31%), Benzene (35.22%), Estragole (4.86%), Anethole (37.85%), 2-pentanone (25.21%), 11-Tricosene (26.58%), 2-Pentanone (5.76%), and Octadecanal (38.26%). Plentiful other compounds were also identified in trace quantities. Fennel nano-emulsion is a more effective option when applied as a foliar spray for controlling gray mold on cucumber fruits under greenhouses conditions without phytotoxicity to growing cucumber plants and be able to use as an environmentally friendly bio-cide.