Urbanization has impacted biodiversity and ecosystems at a global scale. At the same time, it has been recognized as a driver of the physical and emotional gap between humans and nature. The lack of direct contact with nature can have a negative impact on several aspects of human well-being and change knowledge and attitudes of people towards the environment. However, this phenomenon is still poorly understood in megacities outside developed countries. Here, we explore the relationship between ecological knowledge and self-reported well-being in an important urban park in Santiago, Chile. We conducted semi-structured surveys of park users to explore their beliefs, preferences, ecological knowledge of plants and birds, and self-reported well-being. Citizens associated urban parks mainly with "nature, " and particularly with the presence of trees and plants. Trees were recognized as the most relevant elements of urban parks; in turn, birds were ranked as the less relevant. Regarding formal ecological knowledge, respondents correctly identified an average of 2.01 plants and 2.44 birds out of a total of 10 for each taxon, and exotic species were more likely to be recognized. Park users also reported high scores for self-reported well-being. Interestingly, variance of self-reported well-being scores tended to increase at low levels of ecological knowledge of trees, but no significant relationship was detected with knowledge of birds, nor native species. Ecological knowledge of trees was positively related to self-reported well-being. Results suggest that parks can positively contribute to bring people closer to nature in middle-income countries. Improving ecological knowledge can be critical to restore the relationship between humans and nature in megacities.