The Plasmodium falciparum M1-aminopeptidase (PfAM1) is a recognized target in malaria, and is potently but not selectively inhibited by the natural pseudopeptide bestatin. Then, the objective of this work was to identify inhibitors of PfAM1 and of the parasite in vitro growth in a library of thirty three synthetic bestatin-based peptidomimetics. Firstly, we kinetically characterized a 6xHis-tagged recombinant variant of PfAM1 (rPfAM1), expressed in Escherichia coli BL21 and purified by Immobilized Metal Affinity Chromatography, using L-aminoacyl-7-amido-4-methylcoumarin (AMC) fluorogenic substrates. The Zn²⁺ cation is inhibitory at concentrations >1 mM. In agreement with previous reports, the optimal pH is neutral (7.2-7.4) with the 4 tested substrates (Ala-, Leu-, Met- and Arg-AMC). The substrate selectivity matches with that reported for native PfAM1. Equally, the inhibition profile is consistent with a metallo-aminopeptidase. Afterwards, we screened the peptidomimetics for inhibition of rPfAM1 and in cultures of P. falciparum 3D7 strain. Four compounds inhibit rPfAM1 and the in vitro growth of the parasite (IC₅₀ = 10-30 mM; similar to bestatin). They are not cytotoxic in vitro for human erythrocytes and HUVEC cells until 200 mM. These structures competitively inhibit the M1-type aminopeptidase activity in isolated live parasites, suggesting a mechanism for the in vitro antimalarial effect of the peptidomimetics through inhibition of native PfAM1. The most potent compound inhibits the native enzymatic activity in a dose-dependent way, and is selective for rPfAM1 regarding the porcine M1-aminopeptidase (a model of M1-aminopeptidases from mammals). These results support the potentialities of this compound as a leader structure in malaria.