Diplocarpon rosae Wolf is the most common and damaging fungal pathogen in roses. Nationwide, the attack of this pathogenic fungus is very serious in most rose gardens due to rapid disease development that typically leads to leaf yellowing and defoliation. This study aimed to assess the way in which the fungus’s attack affects the chemical composition of Rosa sp. leaves. The research was conducted in the summer of 2023 on ten cultivars of rose grown in the rosary in the ‘Alexandru Buia’ Botanical Garden in Craiova. The influence of the cultivar and harvest time was discussed. Leaf black spot on roses produced by the fungus Diplocarpon rosae Wolf affected plants in all cultivars and the differences between cultivars highlighted the sensitivity of the relationship between plants and pathogens when exposed to some microclimatic environment factors. The correlations of attack degree with tannins, chlorophyll, or alkaloids were significant, negative for tannins (r = −0.189 *) and chlorophyll (r = −0.517 ***) and positive for alkaloids (r = 0.510 ***). Between phenolic compounds, tannins, flavonoids, and chlorophyll, very significant positive correlations were found. Alkaloids established negative and also very significant correlations with phenolic compounds (r = −0.403 ***), tannins (r = −0.339 ***), flavonoids (r = −0.409 ***), and chlorophyll (r = −0.604 ***).