While the pandemic is behind us, the world community faces a global threat of bacterial resistance outbreak. One of the key ways to combat the spread of multi-resistant bacteria is infection prevention and control tactics using modern antiseptic and disinfectant compositions. Herein, we continue the path to unravel the structure–activity relationship (SAR) of potent pyridine-derived biocide class bis-quaternary ammonium compounds (QACs). In this study, twenty dihydroxynaphthalene-derivative bis-QACs were subjected to extensive microbiological analysis on planktonic cells and biofilms of the ESKAPE microorganisms. Among them, hit compounds were superior in their bacteriostatic and bactericidal action to commercial mono-QACs and were comparable to the best bis-QAC antiseptic on the market. SAR analysis indicated that the linker conformation does not significantly affect the activity, though structure symmetry and especially lipophilicity had an influence on antibacterial performance. Furthermore, we delve deeper in investigation of the antimicrobial potential of bis-QACs and conducted a variety of assays, including time–kill kinetics, bacterial resistance formation, cell morphology, and cytotoxicity. Studies showed promising results for compounds 5d and 6d, indicating 2 to 3-fold less cytotoxicity and hemotoxicity compared to commercial QACs. Moreover, SEM imaging revealed that bis-QACs can cause severe membrane damage to S. aureus and P. aeruginosa strains, confirming great potential of novel compounds as antiseptic and disinfectant.