We utilized a series of analogs of D-V13K (a 26-residue amphipathic alpha-helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity for zygomycota fungi decreased with increasing hydrophobicity (D-V13K/A12L/A20L/A23L, denoted D4, the most hydrophobic analog was sixfold less active than D1, the least hydrophobic analog). In contrast, antifungal activity for ascomycota fungi increased with increasing hydrophobicity (D4, the most hydrophobic analog was fivefold more active than D1). Hemolytic activity is dramatically affected by increasing hydrophobicity with peptide D4 being 286-fold more hemolytic than peptide D1. The therapeutic index for peptide D1 is 1569-fold and 62-fold better for zygomycota fungi and ascomycota fungi, respectively, compared with peptide D4. To reduce the hemolytic activity of peptide D4 and improve/maintain the antifungal activity of D4, we substituted another lysine residue in the center of the non-polar face (V16K) to generate D5 (D-V13K/V16K/A12L/A20L/A23L). This analog D5 decreased hemolytic activity by 13-fold, enhanced antifungal activity to zygomycota fungi by 16-fold and improved the therapeutic index by 201-fold compared with D4 and represents a unique approach to control specificity while maintaining high hydrophobicity in the two hydrophobic segments on the non-polar face of D5.