Plum is a stone fruit that stands out for having a short shelf-life because of its high susceptibility to rapid deterioration. Part of this deterioration is explained by fruit overripening. Recently, the role of melatonin in delaying postharvest decay has been investigated but its regulatory function during overripening is still under extensive debate. In this study, to understand physiological events taking place in plums overripening and elucidate the role of melatonin on the postharvest quality of these fruits and its relationship to other plant hormones, Angeleno plums were sprayed with 10−4 M of melatonin solution immediately after harvest. We carried out tissue-specific (mesocarp and exocarp) analysis of total phenols and anthocyanin quantification, as well as the evaluation of different phytohormones by LC-MS/MS and fruit quality parameters. Results showed that during postharvest, endogenous melatonin contents decreased both in the mesocarp and the exocarp of Angeleno plums. Likewise, plum firmness also decreased and a strong correlation was found for this parameter with jasmonic acid (JA) and cytokinins. Conversely, after exogenous melatonin application, endogenous melatonin content increased both in mesocarp and exocarp but it had a differential effect depending on the plum tissue. Indeed, total phenol and anthocyanin contents arose by 21% and 58%, respectively, in the mesocarp after melatonin treatment but no variations were found in the exocarp of Angeleno plums. Hormonal analysis of Angeleno mesocarp also revealed an increase in the JA and its precursor, 12-oxo-phytodienoic acid (OPDA), on the fourth day after melatonin application as well as a positive correlation between melatonin and gibberellin 1 (GA1). These results suggest that melatonin may be acting as a signal molecule increasing phenolic compounds contents through direct regulation and by signaling with other phytohormones. Therefore, this research provides valuable information for understanding the regulatory role of melatonin and its relationship with plant hormones during overripening to contribute to improve the postharvest quality of plums.