The ethanolic root extract of Berberis brevissima afforded a new bisbenzylisoquinoline alkaloid, 13-nitrochondrofoline (2), and two known bisbenzylisoquinoline alkaloids, chondrofoline (1) and curine (4). The acetylation of chondrofoline (1) gave O-acetylchondrofoline (3). The dimeric structures of 1 and 2 were studied through variable-temperature 1H NMR spectroscopy at 25, 40, 60, and 80 °C and conformational analysis, using density functional theory employing the M06-2X functional and the 6-31G* basis set. The in vitro antitrypanosomal activity of compounds 1, 2, 3, and 4 against Trypanosoma brucei showed significant potential with MIC values of 2.6, 2.2, 2.3, and 3.8 μM, respectively. Molecular docking evaluation of alkaloids 1, 2, 3, and 4 against known T. brucei protein targets revealed T. brucei phosphodiesterase B1 to be the preferred target. The docking energies of the alkaloids with Tb6PGL (PDB 3EB9) ranged from -88.8 to -106.0 kJ/mol and was comparable to the cocrystallized ligand, citrate (Edock = -78.3 kJ/mol). It seems reasonable that the curine alkaloids may compete with the natural substrates for these protein targets and serve as leads in designing and developing more potent and selective drugs against T. brucei.