Acanthamoeba keratitis is a sight-threatening infection of the cornea which is extremely challenging to treat. Understanding this organism’s responses during contact lens contact and disinfection could enhance our understanding of how Acanthamoebae colonize contact lens cases, better inform us on contact lens care solution (CLC) efficacy, and help us better understand the efficacy required of CLC products. To explore this gap in knowledge, we used Acanthamoeba ATCC 30461 and ATCC 50370 trophozoites to examine Acanthamoeba behavior during and after CLC disinfection. Amoebae were added to sterile aluminum flow cells and flow cell solutions were changed to Ringer’s solution (control), or one of four CLCs based on biocides (PHMB, PAPB/Polyquad, Polyquad/Aldox, or Polyquad/Alexidine) for 6 h. Each flow cell solution was then changed to axenic culture media (AC6) for 12 h to determine the behavior of amoebae following disinfection. Distance, speed, and displacement were calculated for each organism. As compared to the control of one-quarter Ringer’s solution, each CLC significantly impacted Acanthamoeba motility in both the CLC and AC6 conditions. However, the amoebae challenged with the PHMB CLC traveled a significantly greater total distance than with the other three CLCs, indicating differences in effectiveness between biocides. Furthermore, amoebae regaining motility post-disinfection by CLCs were observed to travel considerable distances and thus could be considered dangerous to ocular health. We determined that while all CLCs produced a substantial or complete cessation of movement vs. the control condition during disinfection, those which relied on the Polyquad biocides were the most effective, and that any amoebae which survived disinfection were able to recover motility. Future examinations of these findings should include direct correlations between motility and viability, and how infectivity and motility may be related.