The increasing prevalence of multidrug-resistant pathogens urged the development of new therapeutic strategies, and antimicrobial peptides (AMPs) have emerged as promising candidates. Indolicidin, a proline-rich AMP, is effective against a wide range of pathogens by penetrating the membrane to disrupt the cytoplasmic membrane or inhibit DNA synthesis. This study investigates the impact of replacing the central proline residue in Indolicidin with glycine (IND-7G), D-proline (IND-7DP), or lysine (IND-7K). Results show that both glycine and D-proline substitutions significantly reduced antimicrobial activity with lower hemolysis than the parent peptide. Besides, the analog having lysine substitution (IND-7K) slightly increased activity against E. coli and C. albicans but reduced potency against S. aureus, E. faecalis, and K. pneumoniae. The hemolytic activity of IND-7K remained comparable to that of Indolicidin. These findings demonstrated the essential role of proline in maintaining the antimicrobial efficacy of Indolicidin.