The purpose of this in vitro study was to compare the effect of intracoronal bleaching agents associated or unassociated with chlorhexidine gel on dentin microhardness. Sixty human maxillary incisor crowns were divided into six groups, and bleaching agents were sealed into the pulp chambers as follows: sodium perborate + water (SPW), sodium perborate + 2% chlorhexidine gel (SP + CHX), sodium perborate + 30% hydrogen peroxide solution (SP + HP), 37% carbamide peroxide gel (CP), 37% carbamide peroxide gel + 2% chlorhexidine gel (CP+CHX), and water (W). After the bleaching procedure, microhardness testing was carried out on the dentin surface at three different levels: inner, middle, and outer dentin. The greatest reduction in microhardness was observed for the SP + HP group. No differences were observed between the SPW and SP + CHX group. The 2% chlorhexidine gel did not adversely affect dentin microhardness when associated with the tested bleaching agents. CHX might be considered as an antimicrobial vehicle during intracoronal bleaching.