The role of topical corticosteroids in management of Acanthamoeba keratitis (AK) remains controversial. Using a rabbit AK model, we investigated whether corticosteroid use is a risk factor of AK. Acanthamoeba (1 × 105/ml) was incubated with two densities of P. aeruginosa (PA; high-PA: 1 × 108/ml, low-PA: 3 × 105/ml) before corneal inoculation. Rabbit corneas were inoculated with Acanthamoeba alone or Acanthamoeba plus PA and administered levofloxacin and betamethasone sodium phosphate (BSP) eye drops for 5 or 7 days. Infected rabbit eyes were evaluated for clinical score and Acanthamoeba by histological examination. Acanthamoeba alone and BSP treatment did not produce keratitis. Corneas inoculated with Acanthamoeba plus low-PA treated immediately with levofloxacin and BSP remained clear with few infiltrates. Corneas inoculated with Acanthamoeba plus low-PA treated with levofloxacin immediately and BSP 12 h later developed severe keratitis. Corneas inoculated with Acanthamoeba plus high-PA treated immediately with levofloxacin and BSP also developed severe keratitis. Acanthamoebae were detected by PAS staining in corneas inoculated with Acanthamoeba plus high-PA treated with levofloxacin and BSP. Topical corticosteroids have the potential to aggravate AK when cornea is infected by Acanthamoeba with a critical number of bacteria or when corticosteroids are given after infection has established by Acanthamoeba with small number of bacteria.