MyristoylCoA:protein N-myristoyltransferase (NMT) covalently attaches the 14-carbon saturated fatty acid myristate, via an amide bond, to the N- terminal glycine residues of a variety of cellular proteins. Genetic studies have shown that NMT is essential for the viability of the principal fungal pathogens which cause systemic infection in immunosuppressed humans and hence is a target for development of fungicidal drugs. We have generated a class of potent peptidomimetic inhibitors of the NMT from one such fungal pathogen, Candida albicans. The N-terminal tetrapeptide from a substrate analog inhibitor, ALYASKL-NH2, was replaced with an ω-aminoalkanoyl moiety having an optimal 11-carbon chain for inhibition (11-aminoundecanoyl-SKL-NH2, 3a, IC50 = 1.2 ± 0.14 μM). A series of replacements for the C-terminal Leu established that residues containing a lipophilic side chain were most effective, with cyclohexylalanine having the greatest potency (3g, IC50 = 0.36 ± 0.06 μM). Removal of the carboxamide moiety led to a metabolically stable dipeptide inhibitor containing an N(cyclohexylethyl)lysinamide (17e, IC50 = 0.11 ± 0.03 μM). Partial rigidification of the flexible aminoundecanoyl chain produced the dipeptide p-(ω-aminohexyl)phenacetyl-L- seryl-L-lysyl-N-(cyclohexylethyl)amide (26b, IC50 = 0.11 ± 0.04 μM). Subsequent incorporation of an α-methyl substituent into 26b provided the dipeptide analog [2-[p-(ω-aminohexyl)phenyl]propionyl]-L-seryl-L-lysyl-N- (cyclohexylethyl)amide, a very potent inhibitor (48, IC50 = 0.043 ± 0.006 μM), which retained the three essential elements required for recognition by the acyl transferase's peptide binding site.