The mussel inspired polydopamine has acquired great relevance in the field of nanomedicines, owing to its incredible physicochemical properties. Polydopamine nanoparticles (PDA NPs) due to their low cytotoxicity, high biocompatibility and ready biodegradation have already been widely investigated in various drug delivery, chemotherapeutic, and diagnostic applications. In addition, owing to its highly reactive nature, it possesses a very high capability for loading drugs and chemotherapeutics. Therefore, the loading efficiency of PDA NPs for an antibiotic i.e., gentamicin (G) has been investigated in this work. For this purpose, an in-situ polymerization method was studied to load the drug into PDA NPs using variable drug: monomer ratios. Scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) confirmed the successful loading of drug within PDA NPs, mainly via hydrogen bonding between the amine groups of gentamicin and the hydroxyl groups of PDA. The loading amount was quantified by liquid chromatography–mass spectrometry (LC-MS) and the highest percentage loading capacity was achieved for G-PDA prepared with drug to monomer ratio of 1:1. Moreover, the gentamicin loaded PDA NPs were tested in a preliminary antibacterial evaluation using the broth microdilution method against both Gram-(+) Staphylococcus aureus and Gram-(−) Pseudomonas aeruginosa microorganisms. The highest loaded G-PDA sample exhibited the lowest minimum inhibitory concentration and minimum bactericidal concentration values. The developed gentamicin loaded PDA is very promising for long term drug release and treating various microbial infections.