The alkaloids demissidine, dihydrosolacongestidine, solasodine and tomatidine were isolated and identified by spectroscopic methods and by comparison with literature data, from the aerial part of Solanum leucocarpum Dunal (Solanaceae) collected at the Regional Natural Park Ucumarí (Colombia). In order to examine topoisomerase I and II activities, the four isolated alkaloids were tested through a mechanism-based bioassay utilizing genetically engineered mutants of yeast Saccharomyces cerevisiae strains RS322N, R52Y and RS321. Dihydrosolacongestidine showed selective activity towards RS321 (249.8 μg/mL) strain rather than RS322N (1542.7 μg/mL), most probably indicating activity through inhibition of DNA topoisomerase II. In the same manner, tomatidine displayed activity through DNA topoisomerase II inhibition. Furthermore, solasodine showed DNA-damaging activity. In addition, the same isolated alkaloids were tested for antibacterial activity by using the agar well diffusion method. Solasodine and dihydrosolacongestidine showed MIC values of 62.5 and 125 μg/mL against Staphylococcus aureus, respectively. The isolation of these alkaloids and their biological activities are reported here for the first time for S. leucocarpum.