Benzalkonium chloride (BAC) is known to cause corneal epithelial damage. In this study we investigated the effect of a BAC solution containing a thickening agent, which enhanced residence time in the eyes, on corneal wound healing using in vivo rat model debrided corneal epithelium. 0.5% or 1.0% methylcellulose (MC), carboxymethylcellulose (CMC) and hydroxypropyl-methylcellulose (HPMC) were used as the thickening agent. The levels of corneal wound healing of rat eyes injected with saline were alone approximately 45.0% at 12 h and 93.6% at 24 h after corneal epithelial abrasion, and healing was almost complete at 36 h. The healing rate in the rat eye treated just with MC, CMC and HPMC was higher than that in those injected with saline. In contrast to the treatment result using only this thickening agent, the healing rate in the eye treated with BAC was lower than that in those injected with saline: the corneal wounds in the BAC-treated eye showed approximately 20% healing at 12 h after abrasion. The injection of 0.02% BAC solution containing MC, CMC and HPMC more significantly delayed the healing than did the injection of 0.02% BAC alone. The results show that the in vivo evaluation method for corneal damage using rat debrided corneal epithelium reflects a toxic change depending upon residence time. These findings provide valuable safety and efficacy information for use in the design of eye drops.