Biofilm formation by pathogenic bacteria is one of the major threats in hospital related infections, hence inhibiting and eradicating biofilms has become a primary target for developing new anti-infection approaches. The present study was aimed to develop novel antibiofilm agents against two Gram-positive bacteria; Staphylococcus aureus (ATCC 43300) and Streptococcus mutans (ATCC 25175) using gold nanomaterials conjugated with 3-(diphenylphosphino)propionic acid (Au-LPa). Gold nanomaterials with different sizes as 2–3 nm small and 9–90 nm (50 nm average size) large were stabilized by LPa via different chemical synthetic strategies. The nanomaterials were fully characterized using atomic force microscope (AFM), transmission electron microscope, ultraviolet–visible absorption spectroscopy, and Fourier transformation infrared spectroscopy. Antibiofilm activity of Au-LPa nanomaterials was tested using LPa alone, Au-LPa and unprotected gold nanomaterials against the both biofilm-producing bacteria. The results showed that LPa alone did not inhibit biofilm formation to a significant extent below 0.025 mM, while conjugation with gold nanomaterials displayed manifold enhanced antibiofilm potential against both strains. Moreover, it was also observed that the antibiofilm potency of the Au-LPa nanomaterials varies with size variations of nanomaterials. AFM analysis of biofilms further complemented the assay results and provided morphological aspects of the antibiofilm action of Au-LPa nanomaterials.