An animal study was carried out on 36 rabbits, whose corneas were inoculated with either Acanthamoeba castellanii alone, Pseudomonas aeruginosa alone or both pathogens together after a period of short (two hours) or long (six hours) co-incubation. The clinical severity of ensuing keratitis was compared between groups daily until day five. Corneas, inoculated with Pseudomonas either alone or in conjunction with Acanthamoeba, were treated with levofloxacin drops three times per day. Corneas inoculated with Acanthamoeba did not develop keratitis. Rabbits inoculated just with Pseudomonas developed mild keratitis and those inoculated with mixture of two pathogens developed significantly more severe keratitis. Most severe disease was present in eyes where Acanthamoeba was pre-incubated with higher concentration of Pseudomonas for a longer period of time. These results suggest that presence of bacteria in specific numbers with adequate co-incubation time is essential for Acanthamoeba to cause clinical keratitis. Further in vivo and in vitro studies with longer follow-up are required.