βCDPEG5 and βCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to βCD. As βCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of βCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO2− overproduction in macrophages, we found that βCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to βCDPEGs and the parent compounds, a similar pattern was observed: the effect of βCDPEG5 on cell viability and cell cycle progression was larger than that of βCDPEG2; PEG2 affected cell viability and cell cycle more than βCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of βCDPEG5 with cell migration was smaller than that of βCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of βCD and PEGs, particularly to yield βCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of βCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from βCD and PEGs.