Female flies (Phorbia phrenione) cross fertilize Epichloë typhina via a specific behaviour in which they transfer spores among self-incompatible fungi. Flies ingest spores while visiting fungi for egg laying. Only 11% of stromata lacking P. phrenione eggs produced perithecia and these perithecia covered an average of only 8% of the stromatal surfaces. In contrast, 71% of stromata that possessed fly eggs produced perithecia, covering 54% of the stromatal surfaces. Immediately following oviposition, flies stereotypically drag their abdomen across the fungus in a spiral pattern while excreting faeces. Transfer of fly faeces to unfertilized stromata resulted in cross fertilization. We observed spiralling patterns of perithecial development on fungal surfaces, signifying the behaviour of the fly results in cross fertilization of the fungus. This is the first documentation of active fertilization in an insect-fungus mutualism.