Purpose: The aim of this in vitro study was to evaluate the influence of preapplication of 2% chlorhexidine gluconate on the immediate microtensile bond strength of a universal adhesive system on dentine subjected to different bonding protocols. Materials and Methods: Twenty human molars were used in this study, and the tooth surface was abraded to expose the dentine. The teeth were randomly divided into four groups according to the surface treatment (n = 5): SBU group: Single Bond Universal without acid etching; SBUPA group: 37% phosphoric acid + Single Bond Universal; SBUCG group: 2% chlorhexidine gluconate + Single Bond Universal; and SBUPACG group: 37% phosphoric acid + 2% chlorhexidine gluconate + Single Bond Universal. The microtensile bond strengths were measured using a microtensile tester 24 h after bonding. The bond strength data were subjected to analysis of variance (ANOVA) and Sheffé's least statistically significant difference test (α = 0.05). Results: No statistically significant differences between the analysed groups were observed (p > 0.05). However, conditioning with phosphoric acid without the action of the chlorhexidine gluconate group resulted in higher numerical values of bond strengths than that for the chlorhexidine gluconate without the acid conditioning group. Conclusion: The preapplication of 2% chlorhexidine gluconate did not reduce the immediate bond strength of the Single Bond Universal adhesive system under different bonding protocols.