The aim of the study was to characterize the chemical composition and biological properties of the essential oil from the plant Lippia citriodora grown in Greece. The essential oil volatiles were analyzed by gas chromatography-mass spectrometry GC-MS indicating citral as the major component. Τhe antimicrobial properties were assayed using the disk diffusion method and the minimum inhibitory and non-inhibitory concentration values were determined. Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Saccharomyces cerevisiae, and Aspergillus niger were sensitive to Lippia citriodora oil, but not Escherichia coli, Salmonella Enteritidis, Salmonella typhimurium, and Pseudomonas fragi. Adversely, all microbes tested were sensitive to citral. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays were used to assess direct antioxidant activity, which proved to be weak for both agents, while comet assay was utilized to study the cytoprotective effects against H₂O₂-induced oxidative damage in Jurkat cells. Interestingly, the oil showed a more profound cytoprotective effect compared to citral. The antiproliferative activity was evaluated in a panel of cancer cell lines using the sulforhodamine B (SRB) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-S-(phenylamino) carbonyl-2-tetrazolium hydroxide (XTT) assays and both agents demonstrated potent antiproliferative activity with citral being more cytotoxic than the oil. Taken together, the essential oil of Lippia citriodora and its major component, citral, exert diverse biological properties worthy of further investigation.