Background. The investigation of volatile oils used in traditional medicine is vital to enhance the quality of healthcare. This study is aimed at screening the antioxidant and antimicrobial properties of Micromeria fruticosa serpyllifolia volatile oils from three different regions in Palestine (north, middle, and south). Methods. Volatile oils of three samples of M. fruticosa serpyllifolia were extracted using the microwave-ultrasonic apparatus. The antioxidant activity of the volatile oils was assessed by inhibition of DPPH free radical. The antimicrobial activity was examined using the broth microdilution method. Assessment of antifungal activity was achieved using the agar dilution method. Results. Screening the biological activity of plant extracts revealed that the sample from Ramallah (middle region) possessed the most potent antioxidant activity with an IC50 value of 0.45 μg/mL. The three samples exhibited broad antimicrobial activity and showed potential antifungal activity. The sample from the southern region showed the highest potency against Shigella sonnei with the lowest reported MIC; the sample from the northern region demonstrated the least potency against clinical isolate of Staphylococcus aureus and “methicillin”-resistant Staphylococcus aureus. Conclusions. The study showed that Micromeria fruticosa serpyllifolia volatile oil samples from different regions in Palestine possess different potential antioxidant and antimicrobial activities that were in line with traditional uses of the plant extracts.