Various functional polydopamine (PDA) nanoparticles have been developed to treat biofilms in recent years; however, in-depth knowledge of how the physicochemical properties of the nanoparticles impact their penetration and accumulation in biofilms is lacking. In this work, PDA nanoparticles of 15, 60, 90, and 200 nm sizes were synthesized and carboxyl, methoxy, and amine groups were introduced to the particle surface to control their surface charges, and then the penetration and accumulation of these PDA nanoparticles in S. aureus biofilms were investigated. The PDA nanoparticles of approximately 60 nm size (PDA60) showed higher penetration and accumulation abilities than nanoparticles of other sizes, and the positively charged amine groups introduced onto the surfaces of PDA60 nanoparticles were more effective than carboxyl or methoxy groups in promoting the interactions of the nanoparticles with the biofilm. The PDA60 nanoparticles with amine surface groups exhibited good photothermal properties, and confocal laser scanning microscopy revealed that they were able to accumulate in the biofilm in significant amounts, which upon irradiation with a laser of 808 nm wavelength showed a high bactericidal rate (97.1%) against the biofilm. The findings of this work provide guidance for the design and development of nanoparticles for antibiofilm application.