Fine particles and their associated compounds such as potentially toxic elements (PTEs) are critical pollutants in ambient air of industrialized urban areas that adversely affect human health. The aim of this study was to determine the pollution, ecological and human health risk assessment of potentially toxic elements in PM2.5 using various indices spatiotemporally, in case of Isfahan city, Iran. Besides, the manuscript objective was to identify an overview and a comprehensive perspective of the potentially toxic elements pollution and risk in PM2.5, affected by anthropogenic and natural source and inversion time using various ecological and health risk assessment indices. The concentrations of 17 PTEs associated with PM2.5 from 40 sampling sites in ambient air of Isfahan city, Iran, were measured. Then their ecological risks (including individual and overall indices) and human health risks (carcinogenic and non-carcinogenic) were assessed. The results of the ecological risk analyses revealed traffic (particularly diesel vehicles) and industrial activities (metal smelting plants, brick and cement factories) together with natural sources (dust storms) affected the risk of PTEs. Among the elements, As, Pb, Cr, and Cd showed the highest health risk, respectively, and only As, Pb, and Cd crossed the carcinogenicity threshold in some sites. Cu, As, and Cd showed the level of highest, average, and potential ecological risk, respectively. Although fall was the most critical season in terms of PTEs ecological risk due to temperature inversion, the human health risk was the highest in summer mainly because of PTEs associated with dust storms. This revealed that health and ecological risk indices vary depending on the source of PM2.5, type and concentration of associated PTEs at different times. The risk trend for different sampling sites was urban > industrial > bus terminal showing the necessity of air pollution control in urban areas with a high population density.