Polyether ionophores, with >120 molecules belonging to this group, represent a class of naturally-occurring compounds that exhibit a broad range of pharmacological properties, including promising activity towards a variety of parasites. In this context, salinomycin (SAL) seems to be interesting, as this ionophore has been found to be active against parasites that are responsible for a number of human and animal diseases. On the other hand, less explored is the investigation into the anti-parasitic activity of SAL derivatives. Recently, we identified C1 amides and esters of SAL and its analogue, C20-oxosalinomycin, as promising structures for trypanocidal drug candidates. In search for novel compounds effective against African trypanosomes, the synthetic access to a completely new series of C20-epi-salinomycin (compound 2) analogues is described in this paper. This series includes products obtained via derivatisation of either the C1 carboxyl or the C20 hydroxyl of 2, but also C1/C20 double modified derivatives. The anti-trypanosomal activity as well as the cytotoxic activity of these analogues were evaluated with bloodstream forms of T. brucei and human myeloid HL-60 cells, respectively. It was found that the C20 single modified derivatives 8, 12, and 18 (C20 decanoate, C20 ethyl carbonate, and C20 allophanate of 2, respectively) were the most active compounds in selectively targeting bloodstream-form trypanosomes, with 50% growth inhibition (GI50) values of 0.027‒0.043 μM and selectivity indices of 165‒353. These results indicate that modification at the C20 position of C20-epi-salinomycin 2 can provide semi-synthetic products with enhanced trypanocidal activity that could be of great value for the development of new drugs to treat African trypanosomiasis.