Chitosan nanoparticles (CNPs) are promising polymeric nanoparticles with exceptional physicochemical, antimicrobial and biological characteristics. The CNPs are preferred for a wide range of applications in the food industry, cosmetics, agriculture, medical, and pharmaceutical fields due to their biocompatibility, biodegradability, eco-friendliness, and non-toxicity. In the current study, a biologically based approach was used to biofabricate CNPs using an aqueous extract ofLavendula angustifolialeaves as a reducing agent. The TEM images show that the CNPs were spherical in shape and ranged in size from 7.24 to 9.77 nm. FTIR analysis revealed the presence of several functional groups, including C–H, C−O, CONH2, NH2, C–OH and C–O–C. The crystalline nature of CNPs is demonstrated by X-ray diffraction. The thermogravimetric analysis revealed that CNPs are thermally stable. The CNPs' surface is positively charged and has a Zeta potential of 10 mV. For optimising CNPs biofabrication, a face-centered central composite design (FCCCD) with 50 experiments was used. The artificial intelligence-based approach was used to analyse, validate, and predict CNPs biofabrication. The optimal conditions for maximum CNPs biofabrication were theoretically determined using the desirability function and experimentally verified. The optimal conditions that maximize CNPs biofabrication (10.11 mg/mL) were determined to be chitosan concentration 0.5%, leaves extract 75%, and initial pH 4.24. The antibiofilm activity of CNPs was evaluated in‑vitro. The results show that 1500 μg/mL of CNPs suppressedP. aeruginosa,S. aureusandC. albicansbiofilm formation by 91.83 ± 1.71%, 55.47 ± 2.12% and 66.4 ± 1.76%; respectively. The promising results of the current study in biofilm inhibition by necrotizing biofilm architecture, reducing its significant constituents and inhibiting microbial cell proliferation encourage their use as natural biosafe and biocompatible anti-adherent coating in antibiofouling membranes, medical bandage/tissues and food packaging materials.