We determined the relative minimal inhibitory and minimal amoebicidal concentrations of chlorhexidine digluconate and polyhexamethylene biguanide for four species ofAcanthamoeba.The amoebae were grown in peptone-glucose-yeast extract broth for 72 h in tissue culture flasks. Either washed trophozoites (∼105) or cysts (∼105) were incubated in the enrichment broth in 96 well microtiter trays. Antimicrobial concentrations of the biguanides were determined from microscopic examinations of methylene blue uptake and from subcultures. In general, killing was time dependent. Minimal amoebicidal concentrations at 24 h ranged from 50 to 100 mg/ml and to as low as 25 mg/ml by 72 h. Trophozoites were killed more rapidly than cysts. Both biguanides had similar levels of activity. A synergistic combination of chlorhexidine and polyhexamethylene biguanide (total concentration 25 mg/ml) was most evident forA. castellaniiandA. polyphaga.Cysts ofA. culbertsoniandA. hatchettistained more rapidly after exposure to the combination of biguanides than to the single biguanides, but there were no statistically significant differences in the final numbers of dead or stained cysts after exposure to the combination or to the single biguanides.