Lepidium sativum L. is a rich source of polyphenols that have huge medicinal and pharmaceutical applications. In the current study, an effective abiotic elicitation strategy was designed for enhanced biosynthesis of polyphenols in callus culture of L. sativum. Callus was exposed to UV-C radiations for different time intervals and various concentrations of melatonin. Secondary metabolites were quantified by using high-performance liquid chromatography (HPLC). Results indicated the total secondary metabolite accumulation of nine quantified compounds was almost three fold higher (36.36 mg/g dry weight (DW)) in melatonin (20 µM) treated cultures, whereas, in response to UV-C (60 min), a 2.5 fold increase (32.33 mg/g DW) was recorded compared to control (13.94 mg/g DW). Metabolic profiling revealed the presence of three major phytochemicals, i.e., chlorogenic acid, kaemferol, and quercetin, in callus culture of L. sativum. Furthermore, antioxidant, antidiabetic, and enzymatic activities of callus cultures were significantly enhanced. Maximum antidiabetic activities (α-glucosidase: 57.84%; α-amylase: 62.66%) were recorded in melatonin (20 µM) treated callus cultures. Overall, melatonin proved to be an effect elicitor compared to UV-C and a positive correlation in these biological activities and phytochemical accumulation was observed. The present study provides a better comparison of both elicitors and their role in the initiation of physiological pathways for enhanced metabolites biosynthesis in vitro callus culture of L. sativum.