The possibility to modify plant metabolic profile of plants and fruit to improve their healthy properties using eco-friendly tools, rather than transgenic approaches, gained interest in the last decades. Ultraviolet-B (UV-B) radiation, at low levels, thanks to its ability to influence plant secondary metabolism, could be successfully used to achieve this goal. However, few studies have been conducted so far on the effects of post-harvest UV-B treatments on fruit metabolomics. The present research, aimed to evaluate the impact of UV-B on peach metabolites profile through nontargeted metabolomics (UHPLC-ESI/QTOF-MS) coupled with multivariate chemometrics, provided evidence that 10 and 60 min of post-harvest UV-B irradiation influenced several classes of metabolites. Most phenolics were downaccumulated 24 h after both UV-B treatments, though, after 36 h, anthocyanins, flavones and dihydroflavonols increased (2.06-, 1.92-, 1.68-fold with 10 min UV-B; 6.65-, 2.53-, 2.05-fold with 60 min UV-B, respectively). UV-B reduced carotenoids and most lipids and increased some biosynthetic intermediates and degradation products, some of them known for their positive role in human health. Among alkaloids, some pteridines accumulated, likely derived from folates degradation, while indole alkaloids decreased. Despite the decrease of some bioprotective metabolites as carotenoids, the UV-B-induced up-accumulation of many antioxidant phenolics after 36 h from the exposure suggests an improvement of the healthy properties of peach fruit and reinforces the potential of UV-B controlled irradiation as a nutraceuticals-increasing tool in fruit.