Cationic biocides, particularly quaternary ammonium compounds (QACs), play a vital role in controlling microbial infections across various industries, healthcare facilities and households. As their widespread use increased drastically in the last few years due to COVID-19, there is growing concern about the development of resistance among microorganisms exposed to cationic biocides. It is crucial to recognize this threat in advance and respond by modifying and replacing the old generation of commercial biocides. Reported here is the pyridinium-based bis-QACs tuning via combination of two simple synthesis approaches to achieve novel biocide's architectures with mixed linkers. The obtained compounds were subjected to a broad bioactivity assay against a panel of 26 microbial pathogens, including multi-resistant bacterial ESKAPEE strains, fungi and biofilms. Novel hit-compounds showed improved antibacterial and antibiofilm action, rapid bacterial eradication within 15-30 min of exposure and 4.5-fold lower hemotoxicity, as well as lower potential for the development of bacterial resistance compared to commercial lead antiseptic octenidine. Highlighted findings and insights will serve as a good basis for further studies of bis-QACs as highly effective biocides.