Medicinal plants are the primary source of resources for practically all traditional healthcare systems. A single herb or mixtures of multiple plants thought to have complementary and/or synergistic effects can be found in herbal medications used in traditional medicine. The aim of this investigation is to identify the bioactive components in the ethanol extracts of eight medicinal plants collected from wild populations in three valleys in the Medina region, Saudi Arabia (Wadi Al-Baidha, Al-Aqiq, and Al-Fora’a). The plants, namely, Asphodelus fistulosus, Commicarpus grandiflorus, Heliotropium arbainense, Pulicaria incise, Rhazya stricta, Rumex vesicarius, Senna alexandrina, and Withania somnifera, were analyzed using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-Mass). The ethanol extract demonstrates the presence of bioactive phenolic compounds, the main components of which were quercetin, luteolin, quercetin, kaempferol-3-glucuronide, and naringenin. The ethanolic extract and its subsequent fractions’ total phenolics and antioxidant activity were quantified. Ethanolic extract had the largest percentage of total phenolic for R. stricta (134 mg/gdw), while R. vesicarius had the highest percentage of total antioxidant (97%). Well-diffusion techniques were used to examine the antimicrobial potential. The findings showed that A. graveolens exhibited significant antibacterial activity against three kinds of bacteria: Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Based on the present results, it is also possible to extend the exams in order to utilize them for medicinal applications.