With a sudden and rapid increase in the spread of antimicrobial resistance, there is an urgent need for the development of novel antimicrobial therapies. Nanoparticles with antimicrobial activity provide advantages such as improved solubility, permeability, stability and selectivity. In this study, a uridine‐scaffold was connected to a pyrene fluorescent unit to obtain novel compound UPy via an economical and straightforward route. Further, fluorescent organic nanoparticles (FONs) were prepared via self assembly of UPy and were found to have antibacterial activity towards Gram positive bacteria. The fluorescent nature of the FONs allowed us to identify a binding partner and propose mechanism of action in vivo. We believe that UPy can be an excellent starting point for the development of novel, potent and selective antimicrobial nanotherapeutics.