Eucalyptus Globulus (EG) is a virtually untapped forest source that belongs to the hardwood family. The objective of this research is to understand the effect of two different isolation techniques, i.e., kraft and organosolv procedures, followed by either acidified sodium chlorite or alkaline hydrogen peroxide treatment on the properties of cellulose and microcrystalline cellulose (MCC) derived from EG. The MCC samples were successfully prepared from cellulose via acid hydrolysis. A comparative study was carried out on the extracted cellulose fibers and MCC samples through deep characterizations of lignocellulosic content, functional groups, crystallinity, thermal properties, and surface morphology. The detailed analyses exhibited that the prepared MCC samples using various approaches are similar to those of commercial MCC. It is revealed that the organosolv treatment followed by acidic bleaching provides the purest MCC with good thermal features, where the obtained cellulose has a glucose content of more than 97% and a degradation temperature of around 343 °C. The present work provides new insight into the effect of various extraction procedures on EG-MCC; these procedures are expected to be used in different industrial applications such as in biorefinery, dietary food, packaging, films, or reinforcement of polymer matrices.