In the context of biology and medicine, nanotechnology encompasses the materials, devices, and systems whose structure and function are relevant for small length scales, from nanometers through microns. The purpose of this study was to compare the microstructures and resultant biocompatibility of three commercially available soft milled cobalt-chromium (Co-Cr) alloys (Ceramill Sintron, CS; Sintermetall, SML; and Soft Metal, SM). Disc-shaped specimens were prepared by milling the soft blanks and subsequent post-sintering. The crystal and microstructures of the three different alloys were studied using optical microscopy, X-ray diffractometry (XRD), energy dispersive X-ray spectroscopy, and electron backscatter diffraction. The amounts of Co, Cr, and molybdenum (Mo) ions released from the alloys were evaluated using inductively coupled plasma-mass spectroscopy. The effect of ion release on the viability of L929 mouse fibroblasts was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The SML alloy showed a finer grain size (approx. 5 μm) and a larger pore size (approx. 5 μm) than the CS and SM alloys, and its XRD pattern exhibited a slightly higher ε phase peak intensity than that of the γ phase. In the CS and SML alloys, the average crystallite sizes of the nano-sized Cr23C6 carbide were 21.6 and 19.3 nm, respectively. The SML alloy showed higher concentrations of Cr and Mo in the grain boundaries than the other two alloys. The SML alloy showed significantly higher Co and Mo ion releases (p < 0.001) and significantly lower cell viability (p < 0.05) than the CS and SM alloys. The combined results of this in vitro study suggest that the three soft milled Co-Cr alloys had different crystal and microstructures and, as a result, different levels of in vitro biocompatibility.