Aim: Acanthamoeba spp. infections are rare but may cause severe keratitis. Boric acid (BA) is widely used in ophthalmic solutions because of its buffering and antiseptic properties. This study aimed to evaluate the direct anti-amoebic activity of BA on Acanthamoeba spp. and its cytotoxic effect on two human cell lines. Material and Methods: An environmental Acanthamoeba spp. isolate was cultivated on a non-nutrient agar medium. The viability of trophozoites and cysts was screened using the trypan blue exclusion method and morphological examination. Chlorhexidine (CHX) was used as the reference drug. Cytotoxic activities of BA and CHX were tested on human neuroblastoma (SH-SY5Y) and immortalized human keratinocytes (HaCaT) with colourimetric methyl thiazolyl tetrazolium (MTT) assay. Results: At 50 mg/mL of BA, the viability of trophozoites decreased to 22.0%±6.0% after 24 hours, whereas no viable trophozoites remained at 1 mg/mL CHX after one hour. Cyst viability was 77.0%±4.4% at the same concentration and time. The cytotoxicity of BA was time- and dose-dependent, with significant decreases at 1000 μg/mL in both cell lines (30.9%±3.1% for SH-SY5Y, 45.4%±0.8% for HaCat). CHX (10 μg/mL) exhibited statistically significant cytotoxicity, 51.5%±1.9% for SH-SY5Y and 51.5%±3.7% for HaCaT cells. Conclusion: This study provided the first data on the direct anti-amoebic activity of BA. BA showed significantly milder anti-amoebic activity compared to CHX. The persistence of viable cysts suggests that contact lens solutions containing BA may carry the risk of Acanthamoeba spp. infection. Further molecular and biochemical studies are needed to clarify this relationship.