Pollution of surface soils by traffic, especially along major highways, can be a significant issue. Numerous studies have demonstrated traffic to be an important source of particulate matter and gas-phase organic air pollutants that produce many types of deleterious effects. This article brings original information about the presence of contaminants with specific mechanisms of action in traffic-influenced soils as determined by bioanalytical approaches and instrumental analyses. The initial phase of the study aimed to compare contamination of soils near highways with those from reference localities, whereas the second phase of the study investigated the influence of traffic pollution in soils at various distances from highways. For the reference areas, forest soils contained greater concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQs; 483 to 2094 pg/g) than did arable soils (96 to 478 pg/g), which represent the relevant reference for the studied soils along highways. The total concentration of TCDD-EQs determined in the in vitro transactivation assay ranged from 225 to 27,700 pg/g in traffic-affected soils. The greatest concentration of TCDD-EQs among the studied sites was observed in soils collected near highway D1, which is the primary thoroughfare in the Czech Republic. The concentrations of TCDD-EQs in roadside soils were the greatest and decreased with increased distance from highways, and this spatial distribution corresponded with the levels of polycyclic aromatic hydrocarbons (PAHs). Soils collected 100 m away from highways in most cases contained concentrations of TCDD-EQs similar to background values. Most TCDD-EQ presence was caused by nonpersistent compounds in soils, with a significant contribution from PAHs as well as other unknown nonpersistent chemicals. Extracts from most soils collected near highways exhibited antiestrogenic and in some cases antiandrogenic activities; for several sites the activity was also detected in soils farther from highways. The presence of TCDD-EQs and antihormonal activity in highway-affected soils points to traffic as a source of polluting compounds having specific effects.