Indiana University-Purdue University Indianapolis (IUPUI) / Purpose: Dental sealant forms a physical barrier to prevent pit and fissure caries; therefore, the retention rate becomes a main factor of the sealant’s effectiveness. Electrospun nylon-6/N6 nanofibers have shown good mechanical properties, such as high tensile strength and fracture toughness. Chitosan/CH has received significant attention due to properties such as antibacterial activity. The purpose of this study was to synthesize and evaluate the effect of incorporating N6 and CH electrospun nanofibers on the physical-mechanical and antibacterial properties of an experimental resin-based sealant. Methods and Materials: Nanofiber synthesis: N6 pellets were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol at a concentration of 10wt%. Practical-grade chitosan was dissolved in trifluoroacetic acid and dichloromethane (60:40 TFA/DCM) at 7 wt%. Electrospinning parameters were optimized in order to fabricate defect-free N6 and chitosan nanofiber mats. Morphological and chemical characterizations were performed by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy, respectively after vacuum drying the mats for 48 h. The average fiber diameter was determined from SEM images by measuring the diameter of 120 fibers using ImageJ software. Experimental Sealant: N6 and CH electrospun mats (3×3cm2) were immersed into a resin mixture of BIS-GMA/TEGDMA. Once no bubbles were seen, the resin-modified N6 and CH mats were put on a glass plate, light-cured (“TRIAD 2000”) for 2 min and then submitted to a cryomilling process to obtain a fine micron-sized powder. Three different filler levels (1 wt%, 2.5 wt%, 5 wt%) were used to prepare the N6 and CH incorporated resin-based sealants. Additionally, a commercially available resin-based sealant and the experimental resin mixture (unfilled) were used as controls. Three-point flexural testing, Vickers microhardness testing, and agar diffusion testing were performed on the experimental sealants and the commercial sealant. Data were analyzed by one-way ANOVA and Fisher's Protected Least Significant Differences Pair-wise comparisons between groups (5%). Results: The average fiber diameter for N6 was found to be 503±304 nm and 595±411 nm for CH. No significant difference was found between fiber diameter (p = 0.0601). FTIR confirmed the characteristic peaks for N6 ((CO-NH and [-(CH2)5-].) and CH (N-H and C2F3O2-). CH-5% group had significantly higher (p = 0.0000) FS (115.3±4.5 MPa) than all other groups. CH-1% and CH-2.5% groups had significantly higher FS than the control (unfilled) (p = 0.0016 and p = 0.0033 respectively); Helioseal Clear (p = 0.0000), and nylon groups. N6-5% had significantly higher flexural strength than Helioseal Clear (p = 0.0013) and N6-2.5% (p = 0.0250). CH-1% had significantly higher hardness values than all other groups, and CH-5% (p = 0.0414) had significantly higher values than N6-2.5%. No antibacterial inhibition was seen in any of the tested groups. Conclusions: CH and N6 nanofibers were successfully prepared via electrospinning and used to modify the experimental resin-based dental sealants. The overall results indicated that CH-containing sealants presented the highest flexural strength and hardness; however, none of the CH groups displayed antimicrobial properties. Further investigation is needed to enhance the physico-mechanical properties of the experimental resin-based sealants using nylon-6 and CH.