<p>The <i>in vitro</i> screening of the antifungal activity in a series of quaternary ammonium salts based on pyridoxine revealed a highly active bis-ammonium compound, <i>N</i>,<i>N</i>′-{[(2,2,8-trimethyl-4<i>H</i>-[1,3]dioxino[4,5-<i>c</i>]pyridine-5,6-diyl]bis(methylene)} bis(<i>N</i>-dodecyl-<i>N</i>,<i>N</i>-dimethylammonium) dichloride (<b>KFU-04</b>), which exhibited pronounced antimycotic activity against a panel of various mycelial and yeast fungal pathogens, in particular, 3 reference strains and 15 clinical isolates, including drug-resistant ones. It was found that <b>KFU-04</b> exerts a rapid dose-dependent effect, allowing to reduce the number of CFU of the pathogen by two to three orders of magnitude within two days, with a significant effect being observed already after 8 h from the start of incubation. In contrast to fluconazole and terbinafine, which are used world-wide to treat onychomycosis (fungal nail infections), <b>KFU-04</b> under the conditions of a model experiment was found to efficiently and dose-dependently inhibit the formation of fungal biofilms of the aggressive clinical isolate <i>C. albicans</i> isolated from nail plates. Using two different types of high-resolution microscopy, confocal laser scanning and electron microscopy, it was shown that the membrane and possibly the cell wall of fungi are among the main targets of compound <b>KFU-04</b>. The described powerful and complex antimicrobial effects were not accompanied by toxicity both when applied externally and when administered systemically: <i>in vivo</i> experiments on rodents demonstrated the high safety of <b>KFU-04</b> (LD₅₀ &gt; 2000 mg kg⁻¹). In general, the obtained experimental results provided strong grounds for continuing studies of compound <b>KFU-04</b> as a potential highly efficient broad-spectrum antifungal agent for combating dangerous infections.</p>