Purpose To compare the therapeutic outcomes of polyhexamethylene biguanide (PHMB) and chlorhexidine for Acanthamoeba keratitis. Design Prospective, double-masked, randomized comparative study. Methods Fifty-six eyes of 55 patients with Acanthamoeba keratitis were randomized to receiving PHMB 0.02% or chlorhexidine 0.02%. Diagnosis was made based on positive culture results (cornea or contact lens case) or on clinical grounds. The primary outcome measure was treatment failure defined as failure to induce a favorable clinical response within two weeks. Secondary outcomes were: 1) recovery of visual acuity (VA), 2) the degree of corneal scarring posttreatment, or 3) the need for penetrating keratoplasty. Results Fifty-one eyes completed the study. Twenty-three eyes received PHMB and 28 received chlorhexidine. Ninety-eight percent were contact lens wearers. Eighteen (78%) PHMB patients were treatment successes compared with 24 (85.7%) chlorhexidine patients ( P = .71). Diagnosis was confirmed by positive corneal culture results in 26 cases (51%). Diagnosis was made within 28 days in 29 cases (56.9%), between one and two months in 13 cases (25.5%), and after more than two months in eight cases (15.7%). Improvement in VA was seen in 13 eyes (56.5%) receiving PHMB vs 20 eyes (71.4%) receiving chlorhexidine. Mild one-quadrant corneal scarring was seen in 43.5% of eyes receiving PHMB and 71.4% of eyes receiving chlorhexidine, whereas moderate corneal scarring in two or three quadrants was seen in 21.7% of eyes receiving PHMB and in 10.7% of eyes receiving chlorhexidine. Five eyes worsened while receiving PHMB vs four eyes worsening while receiving chlorhexidine. Penetrating keratoplasty was required in three eyes from PHMB group and in two eyes from chlorhexidine group. Conclusions Outcomes were similar when using PHMB and chlorhexidine as monotherapy agents in treating Acanthamoeba keratitis.