Fresh bulbs of Stenomesson miniatum, a plant belonging to the Amaryllidaceae family with a poorly investigated phytochemical profile, were traditionally employed by Andean healers to treat tumors and abscesses. The aims of this study were to characterize the extract from the bulbs of S. miniatum and to test its cytotoxic and antibacterial potential. A previous structural study of the major extract constituents was extended to include the minor components by means of 13 C-NMR-based dereplication. Cytotoxic activities were evaluated on A431 human epidermoid carcinoma cells through a metabolic assay and on Jurkat human acute T-leukemia cells through a cell-impermeant fluorescent nuclear probe. Antibacterial assays were carried out against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes by using a standardized broth microdilution method. Eleven known Amaryllidaceae alkaloids were identified together with another compound determined as being an extraction artefact. The alkaloid-enriched extract showed good cytotoxic activity against both tumor cell lines, reaching an IC50 of 3.3 µg/mL against A431 cells and of 10.9 µg/mL against Jurkat cells. Biological assays carried out on single fractions showed that activity can be attributed to the presence of pretazettine and haemanthamine. Conversely, no antibacterial activities were recorded for any of the samples.