This study aimed to test the effectiveness of a solution of chlorhexidine (CHX) and D-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS or TPGS) in the treatment of Acanthamoeba keratitis (AK) via a prospective, interventional case series study. Twenty-nine consecutive patients with AK were enrolled. At baseline, best-corrected visual acuity (BCVA), slit lamp examination, confocal microscopy, and polymerase chain reaction (PCR) were performed. Topical therapy with CHX 0.02% and VE-TPGS 0.2% was administered hourly/24 h for the first day, hourly in the daytime for the next three days, and finally, every two hours in the daytime up to one month. BCVA and ocular inflammation were recorded after two weeks, four weeks, and three months from baseline. Mean logMAR BCVA significantly improved at two weeks (0.78) compared to baseline (1.76), remaining stable over time (0.80 at four weeks, 0.77 at three months). Ocular inflammation improved in 14 eyes at 2 weeks, with further slow improvements in all cases. At three months, no patient had signs of corneal inflammation. The presence of corneal scars was first recorded at the two-week follow-up, with an enlargement at the four-week follow-up. At the three-month follow-up, 19 eyes still showed corneal opacities. In conclusion, the tested solution was shown to be effective for the treatment of AK. Furthermore, it might represent a good first-line treatment.