Understanding the health of urban animals is key for preventing and controlling zoonotic diseases. To achieve this goal, it’s necessary to understand what factors could have an impact on those animals’ health. Therefore, the aim of this article is to compare some health characteristics of opossums (Didelphis virginiana) in urban environments with and without human presence. For this, we trapped opossums in two urbanized areas: one dominated by buildings and one by roads and parking lots (plus two control locations in natural areas). During two periods: normal activities and under lockdown due to COVID-19 conditions (when the presence of people was minimal). We found that in the building area, human presence has almost no effect on the opossums’ health (with only minor changes in erythrocyte counts and tar coverage); however, in the parking lot, the opossum population dropped drastically because of human activities (use of vehicles). This suggests that human presence is not harmful for those animals, but cars are. Therefore, the use of vehicles has a negative impact on opossums’ health and could increase the spreading of zoonotic diseases.