Objective: To investigate the effect of chlorhexidine applications in various forms and concentrations on adhesion and failure modes of metal brackets in vitro. Material and methods: Ninety bovine enamel specimens were allocated to six groups (n=15). Metal brackets were bonded on all specimens after chlorhexidine pre-treatments forming the following groups: (1) untreated specimens (control); (2) 40% varnish (EC40, Biodent BV, Netherlands), remnants removed with brushing mimicking patient cleaning; (3) 40% varnish (EC40), remnants removed with brushing mimicking professional cleaning; (4) 1% varnish (Cervitec Plus, Ivoclar vivadent, Schaan, Liechtenstein), remnants not removed; (5) brushed with% 1 gel (Corsodyl, GlaxoSmithKline, Munchenbuchsee, Germany), remnants not removed; (6) immersed in 0.07% mouthrinse (Corsodyl, GlaxoSmithKline, Munchenbuchsee, Germany), remnant not rinsed. Debonding of brackets was performed using a universal testing machine. Data were analysed using one-way ANOVA and post-hoc Scheffe test. Results: Group 4 performed significantly inferior than all the other groups and the control. Group 4 presented the highest number of adhesive failures at the enamel-resin interface whereas in other groups no failures at adhesive-resin interface was observed. Conclusion: Presence of chlorhexidine varnish prior to bracket bonding adversely affects adhesion. Concentration of chlorhexidine pre-treatment has no influence on shear bond strength.