The development of antimicrobial agents with excellent antimicrobial activity, high thermal stability, and non-toxic properties is an essential demand in food packaging, pharmaceutical, and biomedical applications. In the present study, MgO nanoflakes were synthesized via the co-precipitation method by varying calcination temperatures at 400, 500, and 600 °C. The effect of calcination temperature on the morphology, thermal stability, antimicrobial activity, and cytotoxicity of the MgO nanoflakes was analyzed. All synthesized MgO samples showed a Face-Centered Cubic (FCC) structure. As the calcination temperature increased, the MgO crystallite and particle sizes increased, whereas the surface area decreased. In addition, the MgO synthesized from higher calcination temperatures showed higher thermal stability and crystallinity. The MgO synthesized from the lower calcination temperatures (400 and 500 °C) showed superior antimicrobial activity against (100% R) and (100% R) than that from the higher calcination temperature (600 °C). The cytotoxicity test demonstrates slight cytotoxicity in MgO-400 °C at concentrations ranging from 100 to 200 µg/mL. Interestingly, MgO-500 °C and MgO-600 °C demonstrate biocompatibility and exhibit non-cytotoxic effects, respectively, highlighting their potential for practical applications in active food packaging and biomedical fields.