The opportunistic pathogen, Acanthamoeba castellanii is the causative agent for the sight threatening infection Acanthamoeba keratitis (AK). It is commonly associated with contact lens wearers, and prevalence is increasing at an alarming rate due to an inadequate preventive strategy to protect the lens from this protist. This problem is compounded by the lack of an effective acanthamoebocide, particularly with cysticidal activity in the contact lens solutions. We have used cytotoxicity assays and a variety of biophysical approaches to show that two molecules with tails made of alkyl carbon, alkylphosphocholines (APCs) and quaternary ammonium compounds (QACs) had significant chain-length dependent efficacy against A. castellanii trophozoites, the latter producing death via permeabilization, and DNA complexing. QACs were more effective than APCs and had activity against cysts. Conversely, the QAC with 12 alkyl carbon chain, was non toxic, its presence increased A. castellanii trophozoites biomass and delayed encystation by 96 h. Interestingly, it was unable to induce excystation and increased trophozoite sensitivity to APC16. These results present a mono- and multi-inhibitor management strategy effective against trophozoites and cysts that may be useful for formulating into contact lense cleaning solutions and reducing AK incidence.