The growth in the number of road transport is an integral part of Ukraine's development. At the same time, this is a significant negative factor affecting urban ecosystems. In the paper, the most significant aspects and criteria for their evaluation were identified to assess the negative impact of the transport company on the urban ecosystem. The impact of the Kremenchuk Motor Transport Enterprise on the urban ecosystem of the city of Kremenchuk (Poltava region) was assessed. The main pollutants in the region's air basin are stationary sources. However, the predominant sources of air pollution are emissions from mobile sources. The criteria for assessing the level of environmental safety of a motor transport enterprise were improved. The "Comprehensive assessment of the level of environmental safety of the motor transport enterprise" software tool was developed. It was established that under modern conditions, the level of environmental safety of the motor transport enterprise corresponds to the level of average safety. Comprehensive measures were proposed to ensure the regulatory status of the components of the urban ecosystem and environmental safety on the territory of the Kremenchuk Motor Transport Enterprise.