The medium-sized cities of Brazil are those with populations of between 100,000 and 500,000 inhabitants. These cities are growing rapidly, and are characterized by poor urban planning, a loss of biodiversity and decreasing health prospects for residents. Historically, urbanization in Brazil has been associated with incentives to increase the use of private vehicles for local transport. The air pollution resulting from this “car dependency” has been quantified in some major cities in Brazil, such as the megacity of São Paulo. In this paper, we demonstrate through a bioindication study carried out in the Brazilian city of Rondonópolis, that atmospheric pollution resulting from vehicular emissions is also a measurable reality for medium-sized cities. Most Brazilian cities lack air quality measurement equipment, and we used an inexpensive and easy to apply bioindication assay to measure air pollution, and this approach could be easily implemented in those cities and beyond.