To in vitro evaluate the effects of different chlorhexidine (CHX) concentrations and application methods on the microshear bond strength (µSBS) between dentin and universal adhesives (UAs) used in self-etch mode with different functional monomers. In this study, 300 third molars were randomly assigned into 25 experimental groups based on the combinations of different variables: the UAs and functional monomers (Clearfil Universal Bond Quick [MDP, CU, Kuraray], Tokuyama Universal Bond [3D-SR, TU, Tokuyama], Gluma Bond Universal [MDP-4-META, GU, Kulzer], Prime & Bond Elect [MDP-PENTA, PEU, Dentsply Sirona], and Single Bond Universal [MDP-Vitrebond copolymer, SU, 3 M]), the CHX concentration (2% and 0.2%), and the application mode (with rinsing [R], without rinsing [WR]). Two composite resin samples were prepared on each tooth for the study groups (n = 24). Then, these samples were submitted to a µSBS test after 500 thermal cycles (load of 50 kgF and crosshead speed, 0.5 mm/min). The resulting data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney U tests (α = 0.05). The CU-2%CHX-WR, TU-2%CHX-WR, and GU-2%CHX-WR groups had lower µSBS values than the control groups (CU-control, TU-control, and GU-control) (p < 0.05). Using 2% CHX-WR may reduce the SBS of UAs when they were used in self-etch mode. On the other hand, 0.2% CHX can be used with or without rinsing, since this concentration did not affect the μSBS of any UA in self-etch mode, regardless of the functional monomer. When chlorhexidine is used with proper concentrations and application methods, it will not negatively affect the adhesion of a UA to dentin, even if the UA is used in self-etch mode.