Peptide dendrimers are true polypeptides with a branched architecture. They also represent a cost-effective approach to design peptide antimicrobials by amplifying an ensemble of short peptides consisting of three to five residues. Previously, we showed that a cascade-type of peptide dendrimer D4R consisting of four strands of a tetrapeptide RLYR tethered to a branched Lys-scaffold is as potent as tachyplesin 1, a highly potent and salt-insensitive antimicrobial consisting of 17 amino acids. However, the structure-activity relationships of antimicrobial peptide dendrimers, particularly the scaffold effect on antimicrobial activity, remain poorly understood. Here we compare the scaffold effect of the cascade D4R with four pendant peptide dendrimers, each containing three to five parallel strands of RLYR tetrapeptides organized by either a linear Lys- or Orn-based peptide scaffold. When assayed against ten microorganisms, all four series of pendant dendrimers showed different salt sensitivity and antimicrobial profiles that correlated with their scaffolds. The α- and ε- dendrimers (αK and εK series) organized by Lys-scaffolds were broadly active, salt insensitive and as potent as tachyplesin 1, displaying minimum inhibitory concentrations <1 μM whereas the corresponding α- and δ-dendrimers (αO and δO series) tethered to Orn-scaffolds were substantially less active and highly salt sensitive. NMR studies showed that a Lys-scaffold of D2R can form a reverse turn to support two tetrapeptide as parallel β strands. Molecular dynamic Quantum mechanic simulations of D4R revealed that it can fold as a β structure with the dipeptide Leu-Tyr forming a hydrophobic core, suggesting the ability of the Lys-based scaffolds in organizing parallel peptide strands of RLYR by clustering hydrophobic residues. The importance of a Lys scaffold together with the ability to form a hydrophobic core was demonstrated by comparing D4R with its isomeric dendrimer D4Y (Y=YRRL) containing a scrambled RLYR sequence. Under high-salt conditions, D4Y exhibited greatly reduced antimicrobial potency in five test microorganisms and completely lost its activity in the remaining five. Taken together, our results provide a structural basis for understanding the antimicrobial activities of the Lys-and Orn-scaffolds in designing peptide dendrimers under high-salt conditions, a requirement relevant for developing useful antimicrobials under physiological conditions.