The options for managing Fusarium wilt in greenhouse cucumbers are limited by our poor understanding of the modes of survival and dissemination of the pathogen. This study uses a specific quantitative real-time PCR assay for Fusarium oxysporum f. sp. cucumerinum to investigate the significance of flying insects as aerial vectors of the pathogen in a commercial cucumber greenhouse. Shore flies were more frequently detected (35.5%) carrying F. oxysporum f. sp. cucumerinum than sciarids (25%), with both species carrying between 1 × 102 and 1 × 106 pathogen genome copies/individual. Sciarid and shore flies acquired F. oxysporum f. sp. cucumerinum following exposures to agar cultures of the pathogen of up to 94 h. Light microscopy revealed that spores were carried externally on the bodies of the adult flies. The ability of adult sciarid flies to vector the pathogen from peat-grown diseased cucumber plants and infect healthy cucumber plants was demonstrated in a caged glasshouse trial. An inoculum density trial showed that vascular wilt disease was initiated after inoculation of peat-grown seedlings with as few as 1000 conidia. We conclude that sciarid and shore flies play significant roles as vectors of F. oxysporum f. sp. cucumerinum in greenhouse cucumbers and need to be recognized in developing integrated crop management strategies.