Clavaspirin peptide (CSP) is derived from the pharyngeal tissues of the tunicate Styela clava. The 23-amino acid peptide is histidine-rich and amidated at the N-terminus. CSP possesses low antimicrobial and high hemolytic activity at pH 7.4. Therefore, we designed 4 CSP analogs with substituted hydrophobic amino acids to reduce hydrophobic amino acid interactions. These modifications reduced the aggregation and cytotoxicity of the analogs at pH 7.4. The analogs also showed potent antimicrobial activity by accumulating on bacterial cell surfaces and inducing the lytic mechanism against gram-negative and gram-positive cells at pH 5.5 and 7.4. Moreover, exposure to the CSP-4 analog for up to 29 passages did not induce drug resistance in Staphylococcus aureus. Application of CSP-4 to inflamed skin of hairless mice infected with drug-resistant S. aureus (DRSA) significantly reduced skin infections without damaging dermal collagen or elastin. Topically applied CSP-4 penetrated 25–40 µm in the dermis within 30 min, reducing the levels of Toll-like receptor-2, nuclear factor kappa B (NF-κB), and the pro-inflammatory cytokines tumor necrosis factor- α (TNF-α) and interleukin-1β (IL-1 β). These results suggest that CSP-4 could be a promising topical antimicrobial agent for skin diseases caused by DRSA such as S. aureus CCARM 0027.