Despite senescence-induced chlorophyll depletion in plants has been widely studied, the enzymatic background of this physiologically regulated process still remains highly unclear. The purpose of this study was to determine selected biochemical properties of partially purified fractions of chlorophyllase (Chlase, chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from leaves of three Prunus species: bird cherry (Prunus padus L.), European plum (Prunus domestica L.), and sour cherry (Prunus cerasus L.). Secondarily, this report was aimed at comparing seasonal dynamics of Chlase activity and chlorophyll a (Chl a) content within investigated plant systems. Molecular weight of native Chlase F1 has been estimated at 90 kDa (bird cherry) and approximately 100 kDa (European plum and sour cherry), whereas molecular mass of Chlase F2 varied from 35 kDa (European plum) to 60 kDa (sour cherry). Furthermore, enzyme fractions possessed similar optimal pH values ranging from 7.6 to 8.0. It was found that among a broad panel of tested metal ions, Hg(+2), Fe(+2), and Cu(+2) cations showed the most pronounced inhibitory effect on the activity of Chlase. In contrast, the presence of Mg(+2) ions influenced a subtle stimulation of the enzymatic activity. Importantly, although Chlase activity was negatively correlated with the amount of Chl a in leaves of examined Prunus species, detailed comparative analyses revealed an incidental decrement of enzymatic activity in early or moderately senescing leaves. It provides evidence that foliar Chlase is not the only enzyme involved in autumnal chlorophyll breakdown and further in-depth studies elucidating this catabolic process are required.