With the advent of multi-layered and 3D scaffolds, the understanding of microbiome composition and pathogenic mechanisms within polymicrobial biofilms is continuously evolving. A fundamental component in mediating the microenvironment and bacterial-host communication within the biofilm are bilayered nanoparticles secreted by bacteria, known as bacterial extracellular vesicles (BEVs), which transport key biomolecules including proteins, nucleic acids, and metabolites. Their characteristics and microbiome profiles are yet to be explored in the context of in vitro salivary polymicrobial biofilm. This pilot study aimed to compare the profiles of BEVs from salivary biofilm cultured on a 2D tissue culture plate and 3D melt electrowritten medical-grade polycaprolactone (MEW mPCL) scaffold. BEVs derived from MEW mPCL biofilm exhibited enhanced purity and yield without altered EV morphology and lipopolysaccharide (LPS) content, with enriched BEVs-associated DNA from Capnocytophaga, porphyromonas, and veillonella genus. Moreover, compared to saliva controls, MEW mPCL BEVs showed comparable DNA expression of Tannerella forsythia, and Treponema denticola and significantly higher expression in Porphyromonas gingivalis, Eikenella corrodens and Lactobacillus acidophilus. Together, these findings highlight a more detailed microbial profile with BEVs derived from salivary biofilms cultured on 3D MEW PCL scaffolds, which facilitates an effective in vitro model with a greater resemblance to naturally occurring biofilms.