The present lab-based research aimed to assess the impact of different nanofillers: silver (Ag) chitosan (CH), and arginine nanoparticles (ArgNPs) infused in the eighth-generation universal adhesive (UA) on the antimicrobial potency, micro-tensile bond strength (μTBS), and degree of conversion (DC) bonded to carious-affected dentin (CAD) surface. Forty extracted human molars with Class 1 carious lesions were included. CAD was exposed and samples were allocated into four groups based on the type of adhesive (n = 10): Group 1 (UA), Group 2 (1 wt% AgNPs-UA), Group 3 (1 wt% CHNPs-UA), and Group 4 (1 wt% ArgNPs-UA). Survival rate assessment of inoculated Streptococcus mutans was performed after exposing them to different NPs. Characterization of tested nanoparticles (NPs) was also performed using scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDX). The DC of modified and unmodified eighth-generation adhesive cured and uncured was assessed using Fourier transform infrared spectroscopy (FTIR). Composite was bonded on the CAD and samples underwent artificial aging. Beams were prepared for scanning electron microscopy (SEM) and μTBS analysis followed by failure mode assessment using a stereomicroscope. Statistical analysis was conducted using one-way analysis of variance (ANOVA) and Tukey's post hoc test to compare the means and standard deviations (SDs) between the different experimental groups (p <
0.05). Group 2 (1 wt% AgNPs-UA) samples exhibited the lowest S. mutans survival (0.10 ± 0.02 CFU/mL). Whereas Group 1 (UA) displayed the highest survival of tested bacteria (0.41 ± 0.08 CFU/mL). The maximum score of bond strength of composite to the CAD was detected in Group 2 (1 wt% AgNPs-UA) (21.11 ± 0.59 MPa) samples. However, the μTBS was minimal in Group 1 (UA) (16.18 ± 0.91 MPa). DC between the modified adhesive with 1 wt% AgNPs and the unmodified adhesive was comparable (p >
0.05). The incorporation of 1 wt% AgNPs in eighth-generation adhesives represents a favorable and reasonable alternative to unmodified adhesives when used on caries-affected dentin.