To evaluate the effect of copper and zinc oxide nanoparticles (CuZnONps) added into a universal adhesive on the antimicrobial and bonding properties under a cariogenic challenge (CC). An experimental adhesive containing 0.2% CuNps and 5% ZnONps (0.2/5% CuZnNps), and a control (0% CuZnONps) were applied to enamel-free molars (n = 56), distributed into four groups (n = 14) based on adhesive strategy (etch and rinse [ER] and self-etch [SE]). Restored molars were sectioned into specimens and allocated to immediate evaluation (IM) or to a 42-day in situ (CC) [14 volunteers]. Antimicrobial properties were assessed by CFU counts, SEM, bacterial viability, and mineral loss (ML). Bonding properties were evaluated by microtensile bond strength (µTBS), nanoleakage (NL) and degree of conversion (DC). Data were analyzed using ANOVA and Tukey's post-hoc test (α = 0.05). CuZnONps significantly reduced CFU counts for mutans streptococci (p < 0.003) and total fungi (p < 0.03). SEM showed biofilm with complex morphology. Bacterial viability and ML significantly decrease in CuZnONps groups (p < 0.001; p < 0.0002). After CC, μTBS significantly decreased in ER groups (p < 0.002), while SE CuZnONps showed no significant difference between IM and CC (p > 0.35). Control NL significantly increased after CC (p < 0.001). DC values were unaffected (p = 0.29). Under a cariogenic challenge, a universal adhesive containing CuZnONps demonstrated localized antimicrobial activity and reduced dentin mineral loss and nanoleakage. Importantly, the incorporation of these nanoparticles did not compromise bond strength or the degree of conversion.