This work was conducted to study the chemical composition, antioxidant, antibacterial, and antifungal activities of essential oil and hydrolat from Withania frutescens. The essential oil was extracted by hydrodistillation. The chemical characterization was performed using gas chromatography-mass spectrometry (GC/MS). The antioxidant activity was studied using four different assays (DPPH, TAC, FRAP, and β-carotene bleaching). The antibacterial activity test was carried out on multidrug-resistant bacteria including Gram-negative and Gram-positive strains. Antifungal activity was tested on Candida albicans and Saccharomyces cerevisiae. The yield of essential oil (EO) obtained by hydrodistillation of W. frutescens was 0.31% majorly composed of camphor, α-thujone, carvacrol, and thymol. Regarding the antioxidant activities, the concentration of the sample required to inhibit 50% of radicals (IC50) of EO and hydrolat were 14.031 ± 0.012 and 232.081 ± 3.047 µg/mL (DPPH), 4.618 ± 0.045 and 8.997 ± 0.147 µg/mL (FRAP), 0.091 ± 0.007 and 0.131 ± 0.004 mg AAE/mg (TAC), 74.141 ± 1.040% and 40.850 ± 0.083% (β-carotene), respectively. Concerning the antibacterial activity of essential oil and hydrolat, the minimum inhibitory concentration (MIC) values found were 0.006 ± 0.001 and 6.125 ± 0.541 µg/mL (Escherichia coli 57), 0.003 ± 0.001 and 6.125 ± 0.068 µg/mL (Klebsiella pneumoniae), 0.001 ± 0.0 and 6.125 ± 0.046 µg/mL (Pseudomonas aeruginosa) and 0.012 ± 0.003 and 6.125 ± 0.571 µg/mL (Staphylococcus aureus), respectively. MIC values of essential oil and hydrolat vs. both C. albicans and S. cerevisiae were lower than 1/20,480 µg/mL. Based on the findings obtained, essential oils of Withania frutescens can be used as promising natural agents to fight free radical damage and nosocomial antibiotic-resistant microbes.