Anti-infective agent 12 (Compound A09) is a competitive inhibitor of type I signal peptidase (SPase I) with an IC50 of 4.475 μM and a Kd of 16.3 μM. It disrupts bacterial membranes and has biofilm-clearing capabilities. This agent exhibits potent bactericidal activity against Gram-positive bacteria, with MIC values of 4, 4, 8, and 8 μg/mL for Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, and Streptococcus suis, respectively. Despite its effectiveness against multidrug-resistant strains, it shows weaker activity against Gram-negative bacteria, such as Escherichia coli and Salmonella spp. (MIC > 64 μg/mL). Anti-infective agent 12 has low hemolytic properties and demonstrates significant efficacy in mouse skin infection models. It reduces the risk of resistance emerging from single-target mutations and can be used in the study of multidrug-resistant Gram-positive bacterial infections.