The influence of microstructure, and its changes, on microhardness of the amorphous Fe81Si4B13C2 alloy after thermal treatment at different temperatures from 298 K to 973 K (25 °C to 700 °C) was studied. The as-prepared alloy ribbon containing a small amount of crystalline phases, as well as domains of short-range crystalline ordering embedded in the amorphous matrix, exhibits unexpectedly high microhardness, mostly due to its composition. After thermal treatment above 723 K (450 °C), the alloy samples begin to crystallize, creating a nanocomposite structure involving nanocrystals embedded in an amorphous matrix, leading to an increase in microhardness. Further growth of the nanocrystals, as the heating temperature was increased to 973 K (700 °C), caused the change from nanocomposite structure into a more granulated and porous structure, with a dominant type of interface changing from amorphous/crystal to crystal/crystal, leading to a decrease in microhardness.