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3D in vitro cancer models provide a more physiologically relevant representation of tumor architecture and cell-matrix interactions than conventional monolayer cultures. This study evaluated bacterial nanocellulose (BNC) as a scaffold for the 3D culture of colorectal cancer cells. BNC scaffolds were produced using paraffin spheres as porogens and subsequently characterized in terms of morphology, chemical structure, thermal stability, and mechanical properties. Human colorectal cancer cell lines SW480 and SW620, along with CHO-K1 cells, were exposed to BNC leachates and cultured on 2D and 3D BNC matrices. Cells grown on 2D BNC exhibited typical monolayer proliferation, whereas 3D matrices allowed cell infiltration and volumetric distribution, supporting multicellular organization and the formation of spheroid-like aggregates. Cell viability remained in both leachate and direct scaffold cultures. Genotoxicity assays showed low levels of DNA damage, with most cells displaying no detectable genotoxic effects, and mutagenicity tests revealed no significant differences compared to controls (p > 0.05). Overall, the results demonstrate that 3D BNC supports cell viability, multicellular organization, and genomic safety, highlighting its suitability as a biocompatible scaffold for in vitro 3D colon cancer models.