The antifungal activity of hexanoic acid on the phytopathogen Botrytis cinerea was studied. This chemical inhibited both spore germination and mycelial growth in vitro in a concentration- and pH-dependent manner, and stopped spore germination at a very early stage, preventing germ-tube development. The minimum fungicidal concentration (MFC) for in vitro spore germination was 16 mM. Hexanoic acid also inhibited in vitro mycelial growth of germinated spores at an MFC of 12 mM. Studies performed to characterize the mechanisms underlying the antimicrobial effect of hexanoic acid showed that it alters fungal membrane permeability. In addition, hexanoic acid treatment increased the levels of spermine, spermidine, putrescine and cadaverine in B. cinerea mycelia. Spray application of hexanoic acid at fungicidal concentrations on 4-week-old tomato plants prior to fungal inoculation reduced necrosis diameter by approximately 60%. Application of the same hexanoic acid concentrations on previously infected plants reduced further necrosis expansion by around 30%. The results suggest that this chemical acts as a preventive and curative fungicide. Interestingly, treatments with hexanoic acid at concentrations below the MFC in hydroponic solution prior to fungal inoculation significantly reduced necrosis area. These results suggest an inducer effect of plant responses for hexanoic acid treatments at these concentrations. Hexanoic acid is a good candidate for safe antifungal treatments for the control of B. cinerea, which is responsible for many economic losses on fruits, vegetables and flowers.