Colletotrichum chrysophilum (Cc) (formerly C. fructicola) has been identified as the primary cause of Glomerella Leaf Spot (GLS) in apple plants worldwide. However, Colletotrichum nymphaeae (Cn) was recently reported in Southern Brazil as another pathogen responsible for this disease. Thus, this study aimed to compare the virulence of representative Cc and Cn isolates through assays conducted on seedlings, detached leaves, and potted plants. Additionally, in vitro assessments were made for lipolytic, lipase, and cutinase activities, while monitoring the development of pre-infective structures on apple leaves 48 h post-infection. In contrast to Cc, most Cn isolates were unable to induce typical GLS symptoms on intact tissues. One Cn isolate caused symptoms in seedlings and potted plants, but not on detached leaves. These symptoms mainly comprised atypical lesions, raising questions about the underlying factors. While all isolates induced necrosis in wounded tissues, no association was found between fungal virulence and the activities of lipases and cutinases. Notably, a higher frequency of melanized appressoria was observed in the Cc isolate. This study explores the diversity of Colletotrichum species and its interactions with apple, shedding light on the complexities of symptom development and species-specific adaptations.