In this study, polyaminopropyl biguanide (PAPB) was compared to the molecularly closely related polyhexamethylene biguanide (PHMB) with respect to chemical relationship, antiseptic efficacy and cytotoxicity in vitro. Cytotoxicity for human keratinocytes (HaCaTs) and murine fibroblasts (L929) was determined according to ISO EN 10993-5 for both substances. Antimicrobial efficacy tests were performed via determination of the MBC, quantitative suspension method for substances and investigation of two PAPB-or PHMB-containing dressings against Staphyloccoccus aureus, Escherichia coli and Pseudomonas aeruginosa, according to international standards. Prior mass spectrometry was performed for chemical differentiation of the investigated substances. PHMB showed high toxicity even in low concentrations for both tested cell lines and a high antimicrobial efficacy against S. aureus and E. coli. In the case of PAPB, no or only low cytotoxicity was detected after 72 h, whilst comparable antibacterial features are lacking, as PAPB showed no relevant antimicrobial effects. Even though chemically closely related, PAPB proved to be ineffective in bacterial eradication, whilst PHMB showed a high efficacy. The discovery and establishment of safe and effective alternative antiseptics are important issues for the treatment of infected wounds. In particular, rising bacterial resistances to established agents, as well as ongoing discussions of potential toxic or carcinogenic effects emphasize this necessity. Nevertheless, the presented results highlight that even small changes in the chemical structure of related agents such as PHMB and PAPB can dramatically affect their efficacy and, therefore, need to be carefully distinguished and assessed side by side.