The urban microclimate is gradually changing due to climate change, extreme weather conditions, urbanization, and the heat island effect. In such an altered environment, outdoor thermal comfort can have a strong impact on public health and quality of life in urban areas. In this study, three main urban areas in Serbia were selected: Belgrade (Central Serbia), Novi Sad (Northern Serbia), and Niš (Southern Serbia). The focus was on the temporal assessment of OTC, using the UTCI over a period of 20 years (1999–2018) during different seasons. The main aim is the general estimation of the OTC of Belgrade, Novi Sad, and Niš, in order to gain better insight into the bioclimatic condition, current trends and anomalies that have occurred. The analysis was conducted based on an hourly (7 h, 14 h, and 21 h CET) and “day by day” meteorological data set. Findings show the presence of a growing trend in seasonal UTCI anomalies, especially during summer and spring. In addition, there is a notable increase in the number of days above the defined UTCI thresholds for each season. Average annual UTCIs values also show a positive, rising trend, ranging from 0.50 °C to 1.33 °C. The most significant deviations from the average UTCI values, both seasonal and annual, were recorded in 2000, 2007, 2012, 2015, 2017, and 2018.