Helicobacter pylori is a major infective etiological agent of the upper gastrointestinal tract diseases. The bacterium exhibits resistance to various conventional antibiotics, being usually challenging for eradication. Since there is an urge to consider alternative therapeutic strategies, the aim of the study was to examine selected essential oils of plants belonging to families Cupressaceae (Juniperus communis) and Lamiaceae (Hyssopus officinalis, Salvia officinalis, Melissa officinalis, Lavandula angustifolia, Ocimum basilicum and Thymus serpyllum) against H. pylori, using an improved microdilution broth method. The oils were examined in concentration range from 0.03 to 4 μL/mL. The method comprises Brain-heart infusion broth supplemented with yeast extract, horse serum and IsoVitaleX. After 3 day incubation, an equal volume of double strengthen Christensen's urea was added into each well and incubated for additional 4 h. In wells with present H. pylori, the medium changed color from yellow to purple, allowing MIC determination even without a microtitre plate reader. The microtitre format method is convenient as it is less expensive, easier to perform and requires less amount of an anti-H. pylori agent. The improved method enhances specificity to H. pylori, as fast urease activity is almost an exclusive property of this bacterium. The application of the second step incubation with Christensen's urea decreases the possibility of false positive/negative results due to contaminant growth or commonly poor H. pylori growth. Among the examined oils, J. communis, H. officinalis and O. basilicum were not active with the highest applied concentrations, while the most active was T. serpyllum, with MIC 2.0–4.0 μL/mL. This is the first report on essential oils activity of T. serpyllum and H. officinalis against H. pylori.