This study examined the influence of dimethyl sulfoxide (DMSO) on the microtensile bond strength (μTBS) of universal adhesives to dry or wet dentin in the etch-and-rinse approach. Human third molars were cut to reveal the flat mid-coronal dentin surface. The specimens were randomly divided into 16 groups (n = 8) based on dentin condition (dry or wet), dentin pretreatment (with or without DMSO), and adhesive agents (Scotchbond Universal (SBU), Ambar Universal Bond (AUB), G-Premio Bond (GPB), and Clearfil Universal Bond (CUB). The etched-dentin surfaces were pretreated with a 50% DMSO solution before adhesive application. After 24 h or thermocycling, the resin-dentin beams were tested using the microtensile device. μTBS data were analyzed using a four-way ANOVA (α = 0.05). Scanning electron microscopy (SEM) was used to detect nanoleakage at adhesive interfaces. DMSO increased μTBS to wet and dry dentin of CUB, SBU, and AUB but not GPB. CUB, SBU, and AUB bonded better to wet dentin than dry dentin, but GPB bonded equally well to both. DMSO's nanoleakage effect dependent on dentin moisture and the adhesive agent used.