Botrytis cinerea is a necrotic plant fungus that causes gray mold disease in over 200 crops, including grapevine. Due to its genetic plasticity, this fungus presents strong resistance to many fungicides. Thus, new 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 with white light. Tetra-4-sulfonatophenyl porphyrin tetra-ammonium (TPPS) was tested on B. cinerea using light. 1.5 µM TPPS completely inhibited mycelial growth. TPPS (12.5 µM) was tested on three grapevine clones from Chardonnay, Merlot and Sauvignon, grown in vitro for 2 months. Treated root apparatus of the three backgrounds increased thiol production as a molecular protection against photoactivated TPPS, leading to a normal phenotype as compared with control plantlets. Finally, 2-month-old grapevine leaves were infected with 4-day-old mycelium of B. cinerea pre-incubated or not with TPPS. The pre-treated mycelium was unable to infect the detached 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 suggest a strong potential of photo-treatment against B. cinerea mycelium for future agricultural practices in vineyard or other cultures.