Sclerotinia sclerotiorum is a devastating fungal pathogen that can colonize numerous crops. Despite its economic importance, the regulation of its development and pathogenicity remains poorly understood. From a forward genetic screen in S. sclerotiorum, six UV mutants were identified with loss-of-function mutations in SsATG1, SsATG2, SsATG4, SsATG5, SsATG9, and SsATG26. Functional validation through gene knockouts revealed that each ATG is essential for sclerotia formation, although the morphology of appressoria was not significantly altered in the mutants. Different levels of virulence attenuation were observed among these mutants. Autophagy, monitored using GFP-ATG8, showed dynamic activities during sclerotia development. These findings suggest that macroautophagy and pexophagy contribute to sclerotia maturation and virulence processes. Future work will reveal how autophagy controls target organelle or protein turnover to regulate these processes.