This study aimed to investigate the chemical composition, antioxidant properties, and antimicrobial activity of Tanacetum balsamita L. essential oil. The essential oil was extracted using the water distillation method, and then the total phenolic content (using the Folin-Ciocalteu method), total flavonoid content (using the aluminium chloride colorimetric method), antioxidant activity (using DPPH and ABTS free radical scavenging methods), and antimicrobial activity (using disk diffusion agar, well diffusion agar, minimum inhibitory concentration, and minimum bactericidal concentration methods) were evaluated. The total phenolic and flavonoid contents of the essential oil were found to be 29.50 mg gallic acid equivalent per gram and 14.90 mg quercetin equivalent per gram, respectively. The essential oil exhibited significant antioxidant activity, with the ability to scavenge 50.40% and 53.55% of free radicals DPPH and ABTS, respectively. According to the results of disc diffusion agar method, Bacillus subtilis and Salmonella typhi were the most sensitive and resistant bacterial strains, respectively, with zone of inhibition diameters of 16 mm and 10.50 mm. The zone of inhibition diameters for these bacteria in the well diffusion agar method were obtained as 16.30 mm and 10.80 mm, respectively. The minimum inhibitory concentration and minimum bactericidal concentration for B. subtilis were determined as 4 mg/mL and 32 mg/mL, respectively, and for S. typhi, they were 64 mg/mL and greater than 512 mg/mL, respectively. According to the results, T. balsamita essential oil can be used as a natural antioxidant and antimicrobial agent in food products.