Lippia alba is a plant widely studied due to both chemical diversity and bioactivities related to its ethnobotanical uses. In this work, the composition of the volatile secondary metabolites (volatile fractions/essential oil, EO) of the flower/leaves of L. alba (from northern region of Colombia) was determined by solid phase micro-extraction/distillation-solvent extraction/microwave-hydrodistillation/gas chromatography-mass spectrometry (MWHD/GC-MS), along with some in vitro biological properties (cytotoxicity and acetylcholinesterase enzyme [AChe] inhibition) from leaf EO. Outstanding results were found: (i) cis-piperitone oxide (~13%-46%), germacrene D (~11%-30%), and limonene (~10%-22%) characterized the volatile secondary metabolites from different parts of the plant; (ii) leaf EO showed a moderate hemolytic activity (HC 50 : 580 ± 1 µg/mL), a significant cytotoxicity on lymphocytes (LC 50 : 127 ± 3 µg/mL), a high cytotoxicity on HEp2 cell line (LC 50 : 38 ± 2 µg/mL), and a moderate inhibitory effect on AChE (IC 50 : 28 ± 2 µg/mL). Based on these results, a new chemovar of L. alba is reported (represented by cis-piperitone oxide) along with its promising cytotoxic and AChE inhibiting properties.