Biomolecules of microbial origin are gaining attention for their use in various industries, including cosmetics, due to their broad bioactivities, peculiar properties, and sustainability. This study aimed to develop a novel, eco-friendly nanoemulsion from fungal chitosan hydrochloride (ChC), Pseudomonas aeruginosa biosurfactant (PaB), and grape seed oil (GSO), and to assess its antimicrobial action, biofilm control, and biocompatibility. High-energy emulsification was performed to produce the nanoemulsion (CCh-PaB-GSO), which was characterized by FTIR. Its stability was monitored for 30 days via DLS, zeta potential (ZP), and PDI. The minimum inhibitory concentration (MIC) for cariogenic Streptococcus species, inhibitory fraction concentration (FIC), influence on exopolysaccharide (EPS) quantification produced by bacteria, bacteria’s cell wall hydrophobicity, and biofilm control were determined. Biocompatibility was assessed using the HET-CAM technique by determining the irritation potential. FTIR analysis confirmed the formation the interaction between the substances that compound the nanoemulsion. The CCh-PaB-GSO had nanometric micelles (169.5–203.4 nm), PDI (0.241–0.271), and a positive ZP (+20.25 to +31.94 mV). It showed a consistent MIC (2.0 mg/mL CCh, 0.1 mg/mL PaB, and 3.2 mg/mL GSO) for all tested Streptococcus species and an indifferent interaction effect, FIC (1.32). At sub-MIC, the CCh-PaB-GSO effectively reduced EPS and microbial cell wall hydrophobicity, inhibiting biofilm adhesion. The CCh-PaB-GSO demonstrated biocompatibility with no signs of irritation. In conclusion, the ChC-PaB-GSO system forms an effective and stable nanoemulsion with potential for application as an eco-sustainable and biocompatible product for caries control.