The differences in surface charge of different bacteria can be exploited for their separation by capillary electrophoresis. However, this method of separation of microorganisms is beset with various drawbacks such as adhesion of bacteria to the fused silica surface or cluster formation. To overcome these phenomena we investigated the addition of poly(ethylene oxide) as a focusing agent to the running buffer and used calcium and myoinositol hexakisphosphate as specific ions that interact with the bacterial surface, changing its electrical properties and electrophoretic mobilities. In the present work, we applied CZE to identification of E. coli in infected urine (direct injection) from patients with urinary tract infections and to identification of Helicobacter pylori, which is a gram-negative bacillus responsible for one of the most common infections found in humans worldwide. Helicobacter pylori colonize the stomach and are responsible for severe diseases of the gastric tract, ranging from chronic gastric ulcer to gastric cancer.