Niacinamide is a versatile compound widely used in the personal care industry for its ample skin benefits. As a precursor to nicotinamide adenine dinucleotide (NAD+), essential for ATP production and a substrate for poly-ADP-ribose polymerase-1 (PARP-1), studies have highlighted its roles in DNA repair, cellular stress mechanisms, and anti-aging benefits. Niacinamide was also studied for its antimicrobial activity, particularly in the context of host-infection via host immune response, yet its direct antimicrobial activity and the mechanisms of action remain unclear. Its multifunctionality makes it an appealing bioactive molecule for skincare products as well as a potential preservative solution. This study explores niacinamide’s antimicrobial mode of action against four common cosmetic pathogens. Our findings indicate that niacinamide is causing microbial cell cycle arrest; while cells were found to increase their volume and length under treatment to prepare for cell division, complete separation into two daughter cells was prevented. Fluorescence microscopy revealed expanded chromatin, alongside a decreased RNA expression of the DNA-binding protein gene, dps. Finally, niacinamide was found to directly interact with DNA, hindering successful amplification. These unprecedented findings allowed us to add a newly rationalized preservative facete to the wide range of niacinamide multi-functionality.