Antibacterial resistance is one of the biggest threats to health and economy worldwide. In the present work, we evaluated the antibacterial and antibiofilm properties of silver nanoparticles produced by green synthesis with exopolysaccharides produced by Rhodotorula mucilaginosa UANL-001L, against pathogens of clinical importance. The extraction of exopolysaccharides produced by Rhodotorula mucilaginosa was performed according to a previously described method. Synthesis of the nanobiocomposite was performed by mixing silver nitrate, Acacia rigidula, and Rhodotorula mucilaginosa-produced exopolysaccharides. The newly synthesized nanobiocomposite was lyophilized and kept frozen for further analysis. The characterization of the nanobiocomposite was carried out by UV-visible spectroscopic, Fourier transform infrared analysis and the surface morphology was analyzed by Transmission Electron Microscopy. The antibacterial and antibiofilm activity was tested in 96-well plates for different concentrations of the nanobiocomposite against Escherichia. coli ATCC 11229, Staphylococcus aureus ATCC 6538, and Pseudomonas aeruginosa ATCC 27853. The Rhodotorula mucilaginosa-produced exopolysaccharides were useful in the silver nanoparticle synthesis and the resulting nanobiocomposite had antibacterial and antibiofilm activity against Grampositive and Gram-negative bacteria at lower concentrations than previously reported. Due to these properties, the nanobiocomposite seems to be a promising biocide against pathogens of clinical relevance. In addition, the nanobiocomposite proved to be advantageous in the formulation of hybrid metal-polymer coatings for medical devices or industrial settings.