In the face of rising threat of resistant pathogens, antimicrobial peptides (AMPs) offer a viable alternative to the current challenge due to their broad-spectrum activity. This study focuses on enhancing the efficacy of temporin-SHa derived NST-2 peptide (1), which is known for its antimicrobial and anticancer activities. We synthesized new analogs of 1 using three strategies, i.e. retro analog preparation, lysine addition/substitution, and levofloxacin conjugation. Analogs were tested in term of antibacterial, antifungal, and anticancer activities. Analog 2 corresponding to retro analog of NST-2 was found more active but also more hemolytic, reducing its selectivity index and therapeutic potential. Addition of lysine (in analog 3) and lysine substitution (in analog 7) reduced the hemolytic effect resulting in safer peptides. Conjugation with levofloxacin on lysine side chain (in analogs 4 and 5) decreased the hemolytic effect but unfortunately also the antimicrobial and anticancer activities of the analogs. Oppositely, conjugation with levofloxacin at the N-terminus of the peptide via β-alanine linker (in analogs 6 and 8) increased their antimicrobial and anticancer activity but also their hemolytic effect, resulting in less safe/selective analogs. In conclusion, lysine addition/substitution and levofloxacin conjugation, at least at the N-terminal position through β-alanine linker, were found to enhance the therapeutic potential of retro analogs of NST-2 whereas other modifications decreased the activity or increased the toxicity of the peptides.