Origanum onites (Lamiaceae) is an Eastern Mediterranean plant that is widely used in Turkish traditional medicine. This study aimed to investigate the biochemical composition, antimicrobial activity, and antioxidant potential of O. onites. In this study, the biochemical composition of the O. onites ethanol extract (OOEt) was analyzed using GC-MS. The antimicrobial activity was investigated using a disk diffusion test and determining minimum inhibitory concentrations (MIC) against 30 microorganism strains, including 28 bacteria (some multidrug-resistant) and 2 fungi. Additionally, the antioxidant activity was evaluated using the DPPH method. The main component identified was carvacrol. OOEt demonstrated antimicrobial activity against a wide range of tested microorganism strains. OOEt displayed the highest activity against E. faecium (a Gram-positive bacterium) at 100 µL with a 52 mm inhibition zone. Additionally, P. aeruginosa DSMZ 50071 and P. fluorescens P1, which are Gram-negative bacteria, were the most sensitive strains with a 24 mm inhibition zone in 100 µL of OOEt. The data obtained from A. baumannii (a multidrug-resistant strain) is particularly striking, as higher activity was observed compared to all positive controls. All tested fungal strains showed more effective results than positive controls. The antioxidant activity of OOEt was found to be stronger than that of the positive control, ascorbic acid. This study determined that O. onites has significant antimicrobial and antioxidant potential.