Zingiber officinale Roscoe rhizome plays a vital role in food flavoring as well as utilization in folk medicine. Nepal is categorized among the leading countries of Z. officinale rhizome production and export. Mature Z. officinale rhizome collection is based on the major production sites within Nepal, but we are currently unaware of the chemical composition of essential oil for export. A comparative investigation of Z. officinale rhizome essential oil compositions, collected from 12 different sites of the eastern, mid, and western regions of Nepal, has been carried out. The analysis of essential oils chemical composition by gas chromatographic–mass spectral (GC-MS), enantiomeric composition by chiral gas chromatography–mass spectrometry (CGC-MS), and antimicrobial activity was evaluated. Essential oil yields ranged from 0.28% to 0.34%. The results showed that the essential oils obtained in this study contained α-zingiberene (8.6%-24.1%), camphene (7.2%-12.8%), β-phellandrene (3.8%-10.1%), neral (0.6%-11.8%), geranial (1.0%-17.4%), ar-curcumene (3.0%-10.3%), and β-sesquiphellandrene (3.7%-9.7%). With CGC-MS, the enantiomeric distributions of 21 chiral compounds were determined and showed no contrasting enantiomeric distributions. Two essential oil samples (G3 and G5) possessed good antibacterial activity against Pseudomonas aeruginosa (MIC = 78.1μg/mL) and excellent antifungal activity against Aspergillus niger (MIC = 39.1 μg/mL). Based on the content of α-zingiberene and citral (neral and geranial), samples collected from their respective areas might be used to identify an ideal Z. officinale rhizome production zone in Nepal.