Background: Essential oils (EOs) are one of the most important groups of plant secondary metabolites responsible for their various biological activities. They also have wide applications as natural ingredients in many fields like the food, cosmetic, and medicine industries. This study was conducted to determine the chemical composition, enantiomeric distribution, and in-vitro biological activities of Elsholtzia strobilifera (Benth.) Benth. and E. blanda (Benth.) Benth. essential oils of Lamiaceae from Nepal. Methods: Essential oils obtained from hydro-distillation were analyzed by gas-chromatography mass-spectrometry (GC–MS) for their chemical constituents and the enantiomeric distribution of chiral terpenoids were determined by chiral GC–MS. Microbroth dilution was used to investigate the in-vitro antimicrobial activity, while cytotoxic activities were determined by cell viability using a Cell Counting Kit assay. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging and FRAP (ferric-reducing antioxidant power) assays were used to evaluate the antioxidant activities. Results: A total of 55 compounds were identified in E. strobilifera with pinocarvone (40.7%), β-pinene (10%) and cis-pinocamphone (7.9%) as predominant constituents. While, 41 compounds were identified in the E. blanda EOs, including dihydrotagetone (49.1%), ( Z)-tagetone (15.6%) and ( E)-tagetone (8%) as major components; both essential oils were dominated by oxygenated monoterpenoids. The chiral separation and analyses of 12, E. strobilifera and 7, E. blanda essential oil components were performed, respectively, where (–)-β-caryophyllene and (–)-germacrene D were detected as enantiomerically pure in both oils. Elsholtzia EOs exhibited strong antifungal activity with MIC value of 156.3 µg/mL against Aspergillus niger, whereas, E. strobilifera EO against Staphylococcus epidermidis and E. blanda EO against Staphylococcus aureus showed inhibitory effects with MIC values of 156.3 µg/mL. E. strobilifera EO showed very active in-vitro cytotoxicity against the NIH-3T3 cell line with IC₅₀ of 20.2 µg/mL, whereas E. blanda EO displayed effective cytotoxicity against MCF-7 cell line with IC₅₀ of 33.1 µg/mL. The antioxidant activity of E. strobilifera EO was relatively good with IC₅₀ of 188.8 ± 0.7 µg/mL in DPPH and EC₅₀ value of 348.0 ± 0.1 µg/mL in FRAP, as compared to that of E. blanda EO. Conclusion: Elsholtzia EOs showed prominent biological activities emphasizing their importance as natural ingredients.