Several cities worldwide are studying the replacement of their trolleybus systems with diesel buses or battery electric buses, due to their flexibility and lower operational costs. Diesel buses are considered a major cause of gas emissions in cities, while battery electric buses employ cutting-edge technology, but there is still discussion around the topic due to their technology costs, autonomy, and the sustainability of battery packs. In this study, we evaluated the trolleybus system’s potential for reducing emissions, noise pollution, and greenhouse gases (GHGs) when compared to diesel buses. Furthermore, we compared the trolleybus system with battery electric buses in terms of cost and environmental benefits. To do so, a case study was conducted in São Paulo, Brazil, the largest city in Latin America, which operates the second-highest trolleybus system on the American continent. Our results show that the trolleybus system is a feasible alternative to diesel buses when considering environmental aspects. It can be seen as a complementary service for urban transport systems in the city’s transition to clean energy. Finally, the study implications indicate the need for further investigation of the benefits of in-motion-charge technology to generate flexibility in trolleybus systems, and the involvement of stakeholders in the transition matrix energy process in urban bus systems beyond the direct costs.