This study investigated the effect of over-etching and prolonged application time of a universal adhesive on dentin bond strength. Ninety extracted human molars were ground to dentin and randomly allocated into nine groups (G1–9; n = 10 per group), according to the following acid etching and adhesive application times. In the control group (G1), phosphoric acid etching was performed for 15 s followed by application of the universal adhesive Scotchbond Universal (3M) for 20 s, as per manufacturer’s instructions. In groups G2–5, both the etching and adhesive application times were either halved, doubled, quadrupled, or increased eightfold. In groups G6–9, etching times remained the same as in G2–5 (7.5 s, 30 s, 60 s, and 120 s, respectively), but the adhesive application time was set at 20 s as in the control group (G1). Specimens were then restored with a nanofilled composite material and subjected to microtensile bond strength testing. Bond strength data were statistically analyzed by ANOVA and Tukey’s post-hoc tests (α = 0.05). The relationship of bond strength with etching and adhesive application time was examined using linear regression analysis. Treatment of dentin with halved phosphoric acid etching and adhesive application times (G2) resulted in a significant bond strength decrease compared to the control group (G1) and all other test groups, including the group with halved acid etching, but 20 s of adhesive application time (G6). No significant differences in bond strength were found for groups with multiplied etching times and an adhesive application time of 20 s or more, when compared to the control group (G1). In conclusion, a universal adhesive application time of at least 20 s is recommended when bonding to over-etched dentin.