Magnaporthe oryzae is the most devastating pathogen of rice and the main cause of crop losses worldwide. The successful management of blast disease caused by this fungus is a clear necessity. The synthetic peptide PAF104 has been characterized by its inhibition of M. oryzae appressorium formation on hydrophobic surfaces. Growth and the ability of conidia to germinate was not affected by PAF104, indicating the lack of toxicity on fungal conidia. The addition of the cutin monomer 1,16-hexadecanediol does not interfere with the inhibitory effect of PAF104 on in vitro hydrophobic surfaces. On the other hand, inhibition of appressorium formation by PAF104 was nullified by the exogenous addition of cAMP. Our results suggest that PAF104 affects the Pmk1 pathway by repression of the gene expression of MoMSB2, which encodes a sensing surface protein, and the mitogen-activated protein/extracellular signal-regulated kinase kinase kinase MST11. The pathogenicity of M. oryzae was reduced after PAF104 treatment specifically blocking appressorium formation. Our results support PAF104 as a promising compound to control rice blast disease by blocking a specific target related to appressorium formation, a process essential for infection of rice leaves. Moreover, PAF104 is proposed as a lead compound to develop novel specific fungicides with improved properties.