<p>Euglossini bees are important native pollinators that are highly sensitive to environmental changes. Given that urbanization is one of the most significant drivers of land-use change, this study aimed to analyze the responses of Euglossini bee assemblages to urbanization. For this, Euglossini males were sampled with bait traps along an urbanization gradient quantified using the UrbanizationScore software, between September 2022 and August 2023, in Bahia state, Brazil. Linear Models and non-metric multidimensional scaling (nMDS) ordination were used to analyze the bees’ response to urbanization. A total of 836 individuals were identified, representing 13 species and three genera, with <i>Euglossa cordata</i> (Linnaeus, 1758) and <i>Eulaema nigrita</i> Lepeletier, 1841 being the only species present in urban areas. The kleptoparasitic species <i>Exaerete frontalis</i> (Guérin, 1844) and <i>Exaerete smaragdina</i> (Guérin, 1844) were found exclusively in areas with lower urbanization intensity. Linear Models revealed that more than 80% of the variation in species richness (<i>R</i>² = 0.82), abundance (<i>R</i>² = 0.81), diversity (<i>R</i>² = 0.83), and dominance (<i>R</i>² = 0.81) can be explained by variations in urbanization intensity (<i>p</i> &lt; 0.005). nMDS indicated distinct bee assemblages between urban and semi-natural areas, while the presence of peri-urban points within both ellipses (urban and semi-natural) may reflect the heterogeneous characteristics of these transitional areas. Our findings suggest that urbanization acts as an environmental stressor, modifying the structure and composition of Euglossini bees, leading to a simplification of their assemblages due to the intensity of urbanization.</p>