Aims: The exploration of new strategies for effective microbial control is one of the most significant studies in developing new formulations of antimicrobial agents. The increasing prevalence of microbial threats is a pressing threat to public health. Hence, this study aims to investigate the synergies between combinations of polyhexamethylene biguanide (PHMB) and benzalkonium chloride (BKC) compared to the individual PHMB or BKC as active agents for microbial control. A set of combinations of the active ingredient was tested against two Gram-positive bacteria, Bacillus cereus and Staphylococcus aureus, and two Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. Methodology and results: The antibacterial activity of PHMB and BKC was investigated using the microdilution method to determine their minimum inhibitory concentrations (MIC). The results showed that PHMB was more effective against Gram-positive bacteria, with a significant effect on B. cereus with a MIC value of 31.25 ppm, while having a lesser impact on E. coli (125 ppm). The MIC value of BKC was 15.625 ppm for the Gram-positive bacteria and showed a more prominent effect on Gram-negative bacteria. The MIC values were used as the baseline for formulating PHMB and BKC mixtures. The formulated combinations were evaluated using disc diffusion (DD) and well diffusion (WD) methods. The results indicated that the combined active agents have an efficiency similar to the stand-alone effect. The cytotoxic effect of these compounds was also assessed, and they showed toxic traits towards Vero cells, indicating that these two cationic agents acted additively towards each other. Conclusion, significance and impact of study The combination of PHMB and BKC represented a potential strategy of mixed antimicrobial agents, which could be applied in applications such as healthcare and consumer products.