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Credible assessment methods must be applied to evaluate antiseptics’ in vitro activity reliably. Studies indicate that the medium for biofilm culturing should resemble the conditions present at the site of infection. We cultured <i>S. aureus</i>, <i>S. epidermidis</i>, <i>P. aeruginosa</i>, <i>C. albicans</i>, and <i>E. coli</i> biofilms in IVWM (In Vitro Wound Milieu)—the medium reflecting wound milieu—and were compared to the ones cultured in the laboratory microbiological Mueller–Hinton (MH) medium. We analyzed and compared crucial biofilm characteristics and treated microbes with polyhexamethylene biguanide hydrochloride (PHMB), povidone-iodine (PVP-I), and super-oxidized solution with hypochlorites (SOHs). Biofilm biomass of <i>S. aureus</i> and <i>S. epidermidis</i> was higher in IVWM than in MH medium. Microbes cultured in IVWM exhibited greater metabolic activity and thickness than in MH medium. Biofilm of the majority of microbial species was more resistant to PHMB and PVP-I in the IVWM than in the MH medium. <i>P. aeruginosa</i> displayed a two-fold lower MBEC value of PHMB in the IVWM than in the MH medium. PHMB was more effective in the IVWM than in the MH medium against <i>S. aureus</i> biofilm cultured on a biocellulose carrier (instead of polystyrene). The applied improvement of the standard in vitro methodology allows us to predict the effects of treatment of non-healing wounds with specific antiseptics.