Botrytis cinerea is a necrotic fungal plant pathogen responsible for the gray mold disease on more than 200 crops, including grapevine. Due to its genetic plasticity, this fungus presents a strong resistance to a large spectrum of fungicides. Thus, new fighting strategies against B. cinerea are urgently needed. In this context, antimicrobial photodynamic treatment (APDT) was considered. APDT involves the use of a photosensitizer that generates reactive oxygen species upon illumination. Tetra-4-sulfonatophenyl porphyrin tetra-ammonium (TPPS) was tested on B. cinerea; upon light exposure. 1.5 µM TPPS was shown to completely inhibit mycelial growth. A concentration of 12.5 µM TPPS was tested on three genetic background clones from Chardonnay, Merlot and Sauvignon, grown in vitro for 2 months. Treated root apparatus of the three backgrounds was able to increase thiol production as a molecular protection against photoactivated TPPS, leading to a normal phenotype as compared with control plantlets. Finally, two-month-old grapevine leaves were infected with four-day-old mycelium of B. cinerea pre-incubated or not with TPPS. The pre-treated mycelium was unable to infect the isolated leaves of any of the three grapevine varieties after 72 h growth when subjected to a 16 h photoperiod, contrary to untreated mycelium. These results on fungus, plantlets and Botrytis-grapevine leaves, in contact with a very low concentration of TPPS, suggest a strong potential of photo-treatment against Botrytis mycelium for future agricultural practices in vineyard or other cultures.