Acanthamoeba is an opportunistic pathogen, infamous for its resilience against antiprotozoal armamentaria, disinfectants and harsh environments. It is known to cause keratitis, a blind-causing, painful and difficult to treat corneal infection; Acanthamoeba keratitis (AK) mostly associated with contact lens (CL) wear. In this study, 13 clinical isolates of Acanthamoeba from Hyderabad, India were genotyped and the clinical characteristics associated with AK patients (8 males, 5 females) were analysed. Acanthamoeba were initially isolated from corneal scrapes that were cultured on non-nutrient agar supplemented with heat killed Escherichia coli (ATCC 25922). The genotypes of Acanthamoeba spp. were assessed by PCR using genus specific primers (JDP1 and JDP2) and Sanger sequencing. Most of the isolates (92.3%) belonged to genotype T4 (A. castellanii, A. polyphaga or A. triangularis) except one isolate which was a T12 A. healyi. The mean age of the patients was 32.0 (SD ± 17.4) years (95% CI 22.5 to 43.5 years). Of 13 cases, six (46.2%) cases had risk factor of AK where four patients had ocular trauma and two were CL wearers (15.4%); final visual acuity (VA) of 66.7% patients having either of risk factor was not improved by ≥2 lines. Eleven (84.6%) patients were treated with PHMB and chlorhexidine with three (23.1%) also receiving antibacterials, one case was treated with antibiotics only and other had received steroid and antibiotics. The addition of antibacterial treatment did not affect final visual acuity (p>0.05). The final VA was not improved by ≥2 lines among patients having farming occupation (100%), patients aged ≥33 years (83.3%, 5/6), cases with epithelial defect and corneal ring infiltrate size >4mm (66.7%) and showing symptoms for ≥30 days (60%). Overall, the keratitis-causing Acanthamoeba isolates were mainly of T4 genotype, ocular trauma and CL were the reported risk factor of AK. Aging may be an unmodifiable risk factor of AK and more attention is necessary for patients with severe symptoms such as larger epithelial defect and stromal infiltrate.