The aim of this study was to evaluate the effect of 2% chlorhexidine (CHX) application during the bonding protocol on microshear bond strength of two adhesive systems, after storage in different media. Seventy-two human molars had their crowns cut in half and embedded in PVC cylinders with acrylic resin. The specimens were randomly divided into experimental groups (n=12) according to the adhesive system (Ambar and Single Bond 2), use of CHX in the bonding protocol, and time interval (24 h and 15 days) in the storage media (distilled water, mineral oil and 1% sodium hypochlorite -NaOCl). Adhesive systems were applied in accordance to manufacturers' recommendations, with or without the use of CHX, and resin composite (Z350 XT) cylinders were placed on the hybridized dentin. After photoactivation, the specimens were stored in distilled water, mineral oil and 1% NaOCl for 24 h and 15 days. Microshear bond strength was determined at a crosshead speed of 0.5 mm/min until fracture. The bond strength data were analyzed statistically by 4-way ANOVA and Tukey's test (α=5%). Use of CHX in the bonding protocol did not cause loss of bond strength in any of the evaluated situations, irrespective of time and storage medium. The storage medium had no influence on bond strength values after 15 days when the bond protocol without CHX application was used. However, the use of CHX in the protocol influenced negatively the bond strength values for Single Bond 2 after 15 days storage in distilled water and 1% NaOCl.