Abstract: Biofumigation represents a low-impact alternative to chemical disinfestation aimed atcontrolling soil-borne pathogens. This technique exploits the Brassicales defence system, whichreleases biocidal compounds, mainly isothiocyanates, from glucosinolate hydrolysis bymyrosinase. The aim of our researches is to investigate the interactions of common nonpathogenicsoil fungi with compounds related to biofumigation. A soil isolate of Aspergillusflavus was found to be able to enzymatically convert allyl glucosinolate into low toxicitycompounds, in liquid culture and in sterilised soil. This work is focused on investigating ifA. flavus enzymes are able to compete with Brassica carinata myrosinase during biofumigation,preventing or reducing allyl isothiocyanate formation. In sterilised soil treated with Brassicacarinata seed meal, myrosinase prevailed on A. flavus enzymes and allyl isothiocyanate wasrapidly formed. However its concentration progressively decreased, faster than in the control or inthe presence of another fungal isolate (Trichoderma harzianum). The same was observed in vitro,exposing the fungi to the vapours released by the wetted meal without a direct contact. Thismethod was adopted to screen among several fungal isolates, which showed differentialresponses. The findings indicate that fungal enzymes cannot prevent the isothiocyanate formationduring biofumigation, but could cause a decrease of the active principle concentration, possiblyhampering biofumigation efficacy.