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CoFeO and NiFeO nanoparticles (NPs) represent promising candidates for biomedical applications. However, in these systems, the knowledge over how various physical and chemical parameters influence their cytotoxicity remains limited. In this article, we investigated the effect of different calcination temperatures over cytotoxicity of CoFeO and NiFeO NPs, which were synthesized by a sol-gel proteic approach, toward L929 mouse fibroblastic cells. More specifically, we evaluated and compared CoFeO and NiFeO NPs presenting low crystallinity (that were calcined at 400 and 250 °C, respectively) with their highly crystalline counterparts (that were calcined at 800 °C). We found that the increase in the calcination temperature led to the reduction in the concentration of surface defect sites and/or more Co or Ni atoms located at preferential crystalline sites in both cases. A reduction in the cytotoxicity toward mouse fibroblast L929 cells was observed after calcination at 800 °C. Combining with inductively coupled plasma mass spectrometry data, our results indicate that the calcination temperature can be employed as a facile strategy to reduce the cytotoxicity of CoFeO and NiFeO, in which higher temperatures contributed to the decrease in the dissolution of Co or Ni from the NPs. We believe these results may shed new insights into the various parameters that influence cytotoxicity in ferrite NPs, which may pave the way for their widespread applications in biomedicine.