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Glucosinolates, the main secondary metabolites accumulated in cruciferous flora, have a major impact on fortifying plant immunity against diverse pathogens. Although <i>Botrytis cinerea</i> exhibits varying sensitivity to these compounds, current research has yet to fully understand the intricate mechanisms governing its response to glucosinolates. Different species of the genus <i>Botrytis</i> were exposed to glucosinolate-derived isothiocyanates, revealing that <i>B. fabae</i>, <i>B. deweyae</i>, and <i>B. convolute</i>, species with the <i>mfsG</i> transporter gene (Bcin06g00026) not detected with PCR, were more sensitive to isothiocyanates than <i>Botrytis</i> species containing that gene, such as <i>B. cinerea</i>, <i>B. pseudocinerea</i>, and <i>B. byssoidea</i>. This finding was further corroborated by the inability of species with the <i>mfsG</i> gene not detected with PCR to infect plants with a high concentration of glucosinolate-derived isothiocyanates. These results challenge established correlations, revealing varying aggressiveness on different plant substrates. An expression analysis highlighted the gene’s induction in the presence of isothiocyanate, and a bioinformatic investigation identified homologous genes in other <i>Botrytis</i> species. Our study underscored the importance of advanced biotechnology to help understand these proteins and thus offer innovative solutions for agriculture.