In the current study, indolicidin, a known antimicrobial originally isolated from bovine neutrophils, was modified with respect to hydrophobicity and tryptophan content to maximize bioactivity, and minimize cytotoxicity. Since indolicidin contains five tryptophans (very hydrophobic) of its total 13 amino acids, alanine (mildly hydrophobic) was incrementally substituted in its place to generate five novel derivatives with decreasing hydrophobicity. Antimicrobial testing identified two active derivatives with minimum inhibitory concentrations in the 10⁻⁹ g mL⁻¹ range against Candida albicans, as well as broad-spectrum activity against various other Gram-positive/negative pathogens in the 10⁻³–10⁻⁶ g mL⁻¹ range. Cytotoxicity testing yielded minimum hemolytic concentrations of ∼3 × 10⁻³ g mL⁻¹ for both active derivatives, resulting in hemolytic indices of >1.3 × 10⁶ (peptide Δ45) and 3.6 × 10⁵ (peptide Δ5) (improvements of >33 000-fold and ∼10 000-fold, respectively, compared to indolicidin). The potent antimicrobial activity and low cytotoxicity of these derivatives show promise as potential antibiotics.