Recent studies indicate that Acanthamoeba spp. may play a significant role in kidney dysfunction. The aim of the study was to examine the levels of kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and monocyte chemotactic protein 1 (MCP-1), as well as an activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively) in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. The levels of KIM-1, NGAL, and MCP-1 were analyzed by enzyme-linked immunosorbent assay (ELISA), and the activity of MMPs was determined by gelatin zymography. The elevated KIM-1 level was found in the kidneys of immunocompetent mice at the beginning of Acanthamoeba spp. infection. In the immunosuppressed mice, the KIM-1 level was statistically different. The statistically decreased NGAL level was found in the kidneys of immunocompetent mice compared to the uninfected mice. In the immunocompromised mice, we found statistically significant differences in MCP-1 levels between the uninfected and infected groups. There was an increase in the expression of both MMP-2 and MMP-9 in the kidneys of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. compared to the uninfected mice. The results indicate that KIM-1, NGAL, MCP-1, MMP-2, MMP-9, and MMP-9/NGAL might be promising biomarkers of renal acanthamoebiasis.