In this study, antimicrobial investigations for the efficiently synthesized biocompatible Phloroglucinol Succinic acid (PGSA) dendrimer with anionic surfaces were performed using broth dilution method against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a fungal human pathogen (Candida albicans) to determine the minimal inhibitory concentration (MIC) value. Additionally, fluorescence and UV absorbance spectroscopy techniques were used to monitor the release of intracellular materials from the pathogens owing to anionic dendrimers. The exact binding sites of this dendrimer on these pathogens by molecular modelling studies motivated us to report this nanocarrier as a new antimicrobial agent.