Withania aristata (Aiton) Pauquy, a medicinal plant endemic to North African Sahara, is widely employed in traditional herbal pharmacotherapy. In the present study, the chemical composition, antioxidant, antibacterial, and antifungal potencies of extract from the roots of Withania aristata (Aiton) Pauquy (RWA) against drug-resistant microbes were investigated. Briefly, RWA was obtained by maceration with hydro-ethanol and its compounds were identified by use of high-performance liquid chromatography (HPLC). The antioxidant activity of RWA was determined by use of ferric-reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and total antioxidant capacity (TAC). The evaluation of the antimicrobial potential of RWA was performed against drug-resistant pathogenic microbial strains of clinical importance by use of the disc diffusion agar and microdilution assays. Seven compounds were identified in RWA according to HPLC analysis, including cichoric acid, caffeic acid, apigenin, epicatechin, luteolin, quercetin, and p-catechic acid. RWA had excellent antioxidant potency with calculated values of 14.0 ± 0.8 µg/mL (DPPH), 0.37 ± 0.08 mg/mL (FRAP), 760 ± 10 mg AAE/g (TAC), and 81.4% (β-carotene). RWA demonstrated good antibacterial potential against both Gram-negative and Gram-positive bacteria, with inhibition zone diameters ranging from 15.24 ± 1.31 to 19.51 ± 0.74 mm, while all antibiotics used as drug references were infective, except for Oxacillin against S. aureus. Results of the minimum inhibitory concentration (MIC) assay against bacteria showed that RWA had MIC values ranging from 2.13 to 4.83 mg/mL compared to drug references, which had values ranging from 0.031 ± 0.003 to 0.064 ± 0.009 mg/mL. Similarly, respectable antifungal potency was recorded against the fungal strains with inhibition zone diameters ranging from 25.65 ± 1.14 to 29.00 ± 1.51 mm compared to Fluconazole, used as a drug reference, which had values ranging from 31.69 ± 1.92 to 37.74 ± 1.34 mg/mL. Results of MIC assays against fungi showed that RWA had MIC values ranging from 2.84 ± 0.61 to 5.71 ± 0.54 mg/mL compared to drug references, which had values ranging from 2.52 ± 0.03 to 3.21 ± 0.04 mg/mL. According to these outcomes, RWA is considered a promising source of chemical compounds with potent biological properties that can be beneficial as natural antioxidants and formulate a valuable weapon in the fight against a broad spectrum of pathogenic microbes.