The current study was conducted to characterize the vinegar extract of Nigella sativa and evaluate its biological activities using in vitro and in vivo studies. The N. sativa extract (NSE) was prepared by macerating seeds in a mixture of water and synthetic vinegar (1:10). The antioxidant potential of NSE was assessed, revealing high total phenolic contents (431.66 mg/100 g), total flavonoid contents (73.45 mg/100 g), total antioxidant capacity (118.55 mg/100 g), and strong radical scavenging potential (89.67%). The anticancer activity of NSE showed insignificant cytotoxic effects on HepG2 liver cell lines (96.61 ± 3.00). An in vivo animal trial was performed, and NSE significantly (p