The subject of the article was the chemical analysis of gasoline and exhaust gas samples taken from an urban two-wheeled vehicle. The main aim of the work was to identify chemical compounds emitted by a group of urban two-wheeled vehicles depending on the engine’s operating parameters. First, engine operating parameters and driving parameters of three urban two-wheeled vehicles were measured in real operating conditions. Based on the averaged results, engine operating points were determined for exhaust gas samples that were collected into Tedlar bags. The exhaust gas composition of individual chemical substances obtained in the chromatographic separation process were subjected to a detailed analysis relating the engine operating point with their emission rate, with each individual component being assessed in terms of its impact on human health. The obtained qualitative analysis results indicated the presence of alkenes, alkanes, aliphatic aldehydes, and aromatic and cyclic hydrocarbons (cycloalkanes) in the tested samples. The experiments provided a variety of conclusions relating to the operating parameters of a two-wheeler engine. Qualitative assessment of exhaust samples showed that a two-wheeled vehicle was characterized by the most varying composition of BTX aromatic hydrocarbons derivatives, which are particularly dangerous to human health and life. Therefore, the authors suggest that in the future, approval procedures regarding toxic emissions should be extended to include chromatographic tests. The presented results are an extension of previous studies on toxic emissions from urban two-wheeled vehicles in real operating conditions that were published in other journals.