Earth structures have a significant sustainable impact on regulating indoor environmental qualities. Yet, using soil materials can lead to fungal growth, impacting occupant health and structural stability. This study investigates the susceptibility of earth-based construction materials with cement, limestone, and acrylic-based additives to fungal growth. Laboratory tests were conducted on mixtures under conditions found in inhabited buildings in hot–arid regions. The proposed methodology was based on a 7-week artificial incubation of fungi obtained from moldy walls through regulating the room temperature to fall between 18 °C and 19 °C and a controlled humidity level of around 45%. These conditions were adopted according to the readings monitored in typical buildings in the study area. The results showed that fungal growth was evident on the surface of mixtures, including higher percentages of soil and lower percentages of additives. Mixtures comprising 50% soil, 15% acrylic-based additive, 15% quicklime, and 20% cement supported the least fungal growth, presenting the best choice as a sustainable, efficient replacement. Visual observation followed by microscopic examination ensured the results. Furthermore, results of an environmental post-occupancy evaluation of a constructed rammed earth building using the optimized mixture showed no signs of fungal proliferation on the inner walls afterward.