The threat of antimicrobial-resistant bacteria is ever increasing and over the past-decades development of novel therapeutic counter measurements have virtually come to a halt. This circumstance calls for interdisciplinary approaches to design, evaluate and validate the mode of action of novel antibacterial compounds. Hereby, carbosilane dendritic systems that exhibit antimicrobial properties have the potential to serve as synthetic and rationally designed molecules for therapeutic use. The bow-tie type topology of BDTL049 was recently investigated against the Gram-positive model organism Bacillus subtilis, revealing strong bactericidal properties. In this study, we follow up on open questions concerning the usability of BDTL049. For this, we synthesized a fluorescent-labeled version of BDTL049 that maintained all antimicrobial features to unravel the interaction of the compound and bacterial membrane. Subsequently, we highlight the bacterial sensitivity against BDTL049 by performing a mutational study of known resistance determinants. Finally, we address the cytotoxicity of the compound in human cells, unexpectedly revealing a high sensitivity of the eukaryotic cells upon BDTL049 exposure. The insights presented here further elaborate on the unique features of BDTL049 as a promising candidate as an antimicrobial agent while not precluding that further rounds of rational designing are needed to decrease cytotoxicity to ultimately pave the way for synthetic antibiotics toward clinical applicability.