Anuran skin secretions are rich sources of bioactive metabolites whose composition may vary according to environmental conditions. In this study, skin secretions from two populations of Dendropsophus columbianus exposed to contrasting environmental conditions (Clarete and Las Guacas, Popayán, Cauca, Colombia) were evaluated for antifungal activity against Hemileia vastatrix, the causal agent of coffee leaf rust. Secretion from the Clarete population showed no significant inhibition of urediniospore germination (p = 0.9999). In contrast, secretion from Las Guacas, a polluted and poorly conserved environment, exhibited significant dose-dependent antifungal activity (p = 0.0004–<0.0001). Chromatographic profiles from Las Guacas were more complex than those from Clarete. Two fractions, designated 5* and 7*, reduced urediniospore germination to 17.6% and 9.6%, respectively, compared to the negative control (75.37%; p < 0.0001). LC-MS analysis detected low-molecular-weight singly charged compounds (400–1500 m/z) consistent with alkaloid-like secondary metabolites. Although no definitive structural identification was achieved, these findings highlight the chemical plasticity and biotechnological potential of D. columbianus for the sustainable control of coffee leaf rust.