The aim of this study was to investigate the antimicrobial activity of bisquaternary ammonium compounds which contain two quinuclidine rings mutually bound by alkyl chains. The length of the alkyl chains is 8, 10 or 12 carbon atoms linked to the nitrogen atom of quinuclidinyl-3ol (BIS-QOH-8, BIS-QOH-10, BIS-QOH-12). The antimicrobial activity of the tested compounds depends on the length of the chain and increases with the increase in the number of carbon atoms. The best effect was observed against fungi, including opportunistic pathogenic yeast C. albicans and widespread molds that cause food degradation, P. citrinum and A. niger. Given that data on antifungal activity of compounds structurally similar to those in the available scientific literature are scarse, these results represent an additional evidence of a wide biological activity and guidelines for future investigation of quaternary ammonium compounds. During this study, the better effect of the derivatives on gram-positive compared to gram-negative bacteria was observed, which might be related whit their cell wall structure. Inhibition of biofilm S. aureus ATCC 29213 at a different concentration range of the most efficient derivative with the longest chain showed a reduction in biofilm even at the subinhibitory concentrations. These data confirm a wide spectrum activity of the quaternary ammonium compounds and their potential for application in medicine and pharmacology, which is of interest to the scientific public and society in general.