The present study attempts to examine the research gap in terms of comparing the environmental impact of trolleybuses and diesel buses in the conditions of a country with an unfavourable energy mix. The analysed example concerns the trolleybus transport system in Gdynia, in northern Poland, which also partially serves the neighbouring city of Sopot. In the last few years, two bus lines have been electrified with trolleybuses in the In-Motion-Charging technology, which enables operation on sections without an overhead network. Using the actual operational data, a comparative analysis of the emissivity of diesel buses and trolleybuses used on the same lines in an identical operating regime was conducted. Moreover, an attempt was made to estimate the damage costs of the emission of air pollutants for the above-mentioned means of transport. Research has shown that trolleybuses significantly help to reduce emissions of nitrogen oxides, non-methane volatile organic compounds and particulate matter, while increasing sulphur dioxide emissions on the served lines. They also generate lower specific emissions of carbon dioxide compared to diesel buses. However, taking into account the differences in the number of seats in these vehicles, the length of routes resulting from a need to provide access to the necessary infrastructure and the total amount of kilometres covered on a given route, they may cause higher emissions per year and per the product life cycle than diesel buses. This is related to the unfavourable structure of energy production in Poland, which is dominated by coal sources. The research results clearly show that the use of trolleybuses in public transport contributes to a reduction of the damage costs of the emission of pollutants that amount to approximately EUR (€) 30,000–60,000 per year for the analysed lines.