Allosteric inhibitors regulate enzyme activity from remote and usually specific pockets. As they promise an avenue for less toxic and safer drugs, the identification and characterization of allosteric inhibitors has gained great academic and biomedical interest in recent years. Research on falcipain-2 (FP-2), the major papain-like cysteine hemoglobinase of Plasmodium falciparum, might benefit from this strategy to overcome the low selectivity against human cathepsins shown by active site-directed inhibitors. Encouraged by our previous finding that methacycline inhibits FP-2 noncompetitively, here we assessed other five tetracycline derivatives against this target and characterized their inhibition mechanism. As previously shown for methacycline, tetracycline derivatives inhibited FP-2 in a noncompetitive fashion, with Ki values ranging from 121 to 190 μM. A possible binding to the S' side of the FP-2 active site, similar to that described by X-ray crystallography (PDB: 6SSZ) for the noncompetitive inhibitor E-chalcone 48 (EC48), was experimentally discarded by kinetic analysis using a large peptidyl substrate spanning the whole active site. By combining lengthy molecular dynamics (MD) simulations that allowed methacycline to diffuse from solution to different FP-2 surface regions and free energy calculations, we predicted the most likely binding mode of the ligand. Of note, the proposed binding pose explains the low differences in Ki values observed for the tested tetracycline derivatives and the calculated binding free energies match the experimental values. Overall, this study has implications for the design of novel allosteric inhibitors against FP-2 and sets the basis for further optimization of the tetracycline scaffold to produce more potent and selective inhibitors.