​In this research, the chemical make-up and antibacterial activities of Ruta graveolens callus, in vitro and ex vitro extracts were examined. In order to initiate the callus formation, leaf-disc explants were cultured on Murashige and Skoog (MS) media supplemented with 4.0 mg/L α-naphthaleneacetic acid (NAA). A frequent subculture on MS media containing 1.0 mg/L 2,4- Dichlorophenoxyacetic acid (2,4-D) was then used to sustain callus tissue. For root induction, MS media having different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and NAA (0.0, 0.4, 0.8, 1.2, 1.6, 1.8 and 2.0 mg/L) were used.. Five bacterial species, including two Gram-negative bacteria (Escherichia coli and Salmonella typhimurium), three Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus and Micrococcus latus), and four fungal species (Alternaria solani, Sclerotinia sclerotiorum, Fusarium oxosporium and Verticillium dahlia) were tested for the antimicrobial effects of methanolic and ethanolic crude callus, in vitro and ex vitro extracts of R. graveolens. All R. graveolens extracts tested at various concentrations exhibited antimicrobial action against all bacterial species; however, no antifungal activity was identified against the selected fungus species with the exception of Fusarium oxosporum. R. graveolens has the potential to be a standard source for the pharmaceutical industry development of antibacterial and antifungal medicines.​