Helicobacter pylori living in the human stomach release volatile organic compounds (VOCs) that can be detected in expired air. The aim of the study was the application of breath analysis for bacteria detection. It was accomplished by determination of VOCs characteristic for patients with H. pylori and the analysis of gases released by bacteria in suspension. Solid-phase microextraction was applied as a selective technique for preconcentration and isolation of analytes. Gas chromatography coupled with mass spectrometry was used for the separation and identification of volatile analytes in breath samples and bacterial headspace. For data calculation and processing, discriminant and factor analyses were used. Endogenous substances such as isobutane, 2-butanone and ethyl acetate were detected in the breath of persons with H. pylori in the stomach and in the gaseous mixture released by the bacteria strain but they were not identified in the breath of healthy volunteers. The canonical analysis of discrimination functions showed a strong difference between the three examined groups. Knowledge of substances emitted by H. pylori with the application of an optimized breath analysis method might become a very useful tool for noninvasive detection of this bacterium.