The identification of bacteria by using conventional microbiological techniques can be very time-consuming and circumstantial. In contrast, the headspace screening of bacterial cultures by analyzing their emitted volatile compounds using mass spectrometry might provide a novel approach in diagnostic microbiology. In the present study different strains of Escherichia coli, Klebsiella, Citrobacter, Pseudomonas aeruginosa, Staphylococcus aureus, and Helicobacter pylori were investigated. The volatile compounds emitted by these bacteria in vitro were analyzed using proton-transfer-reaction mass spectrometry, which allows rapid and sensitive measurement. The detected patterns of volatile compounds produced by the investigated bacteria were compared and substantial differences regarding both quantity and quality were observed. In conclusion, the present study is the first to describe headspace screening of bacterial cultures as a potential diagnostic approach in medical microbiology.