The assessment of forest biomass has been a focus of research, aiming to understand matter–energy relationships in forest ecosystems and address forestry practice issues. In recent decades there has been increased societal interest in rational forest resource exploitation, necessitating accurate biomass estimation. In Greece there has been limited efforts in estimating forest biomass, and the current study focuses on habitat type 9340, “Quercus ilex and Quercus rotundifolia forests,” located in the protected areas “GR1420004–Karla–Mavrovouni–Kefalovryso Velestinou–Neochori” and “GR1430001–Oros Pilio and Paraktia Thalassia Zoni (Mount Pilion and Coastal Sea Zone)” in the Natura 2000 network. The habitat falls within the thermo-Mediterranean zone and the study aims to estimate the biomass and carbon storage to contribute to sustainable EU forest strategies. Due to resource limitations, a generalized allometric equation was proposed as an alternative to traditional biomass estimation methods. The above-ground biomass per hectare was estimated, ranging from 16.10 to 205.27 Mg ha−1 (mean 61.91 Mg ha−1). Furthermore, two approaches were used to estimate the total biomass in the habitat: regional averages based on spatial distribution and spatial interpolation using a geographic information system. The total estimated biomass for habitat 9340 is 183,505 Mg, with the carbon storage in standing dry biomass amounting to 83,725.25 Mg. Despite the absence of sampled biomass specimens, this study combines robust statistical techniques with published empirical values to provide a solid framework for estimating assimilated CO2. Sequestered CO2 in the study area is estimated at 306,992.58 Mg. Therefore, the significant role of Quercus ilex L. in carbon storage in Mediterranean forest ecosystems is highlighted by sequestering a substantial amount of CO2.