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Concern about microbial tolerance and resistance to established antimicrobials drives research into alternatives for local antiseptic wound treatment. Precise efficacy profiles are thereby important in the evaluation of potential alternative antimicrobials, and protein interference (“protein error”) is a key factor. Here, the antimicrobial efficacy of cetylpyridinium chloride (CPC) and miramistin (MST) was compared to the established antimicrobials octenidine (OCT), povidon-iodine (PVP-I), polyhexamethylene-biguanide (PHMB) and chlorhexidine (CHX). Efficacy was evaluated after 0.5, 1, 3, 5 and 10 min against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecium and Candida albicans using an in vitro quantitative suspension method (based on DIN EN 13727). To investigate protein interference, 0.3% or 3% bovine albumin was used as the challenge. OCT and PVP-I demonstrated a significant efficacy within 0.5 min, regardless of the microbial organism and protein challenge (p < 0.01). CPC and MST showed no inferiority in efficacy, with only MST needing up to 3 min to achieve the same microbial reduction. PHMB and CHX also achieved significant reduction rates over the tested time-course, yet demonstrated a necessity for prolonged exposure (up to 10 min) for comparable reduction. A protein interference was predominantly observed for PHMB against S. aureus, but without statistically significant differences in antimicrobial efficacy between the 0.3% and 3% protein challenges. All other tested agents showed no relevant interference with the presence of protein. CPC and MST proved to be non-inferior to established wound antiseptics agents in vitro. In fact, CPC showed a more efficient reduction than PHMB and CHX despite there being an introduced protein challenge. Both agents demonstrated no significant “protein error” under challenging conditions (3% albumin), posing them as valid potential candidates for alternative antimicrobials in wound management.