Food fortification is defined as the addition of nutrients at levels higher than those found in the original food to improve nutritional quality. Oyster mushrooms generally contain low calcium. This present study focused on the Ca absorption efficacy of Pleurotus florida using eggshell powder, oyster shell powder and agricultural lime. Various concentrations (0%, 2%, 4%, 6%, 8% and 10%) of different calcium sources were added to the rice straw – based formulation. Duration of days from inoculation to mycelial colonization, primordial formation, fruiting body development were evaluated. Furthermore, the effect of Ca sources on the total yield and biological efficiency were also noted. Results revealed that addition of Ca sources on the formulated substrates generally lengthens time of mycelial ramification, primordial formation and fruiting body development. ESP-supplemented substrate generally increased the size of the cap but not greatly affected the stipe length and diameter of the fruiting bodies. On the other hand, the influence of addition of OSP increases cap size but decreases stipe length and diameter. Finally, for the incorporation of AGL, it was observed that size of cap of the fruiting bodies generally decreased in size, shortened the stipe and influence of AGL to the stipe diameter may vary depending on the concentration. Moreover, Ca supplementation increases the yield, biological efficiency and Ca content of P. florida. The highest yield from the substrate supplemented with eggshell powder was recorded at 8% concentration with 194.80g and biological efficiency of 36.26%. Substrate with 8% oyster shell powder resulted with the highest yield of 176.80g with biological efficiency of 33.92%. For agricultural lime supplemented substrate, the highest yield was observed in 6% concentration with 186.40g and biological efficiency of 35.51%. Optimum Ca content absorbed by P. florida was recorded in 6% ESP (140mg/100g dried sample), 10% OSP (105mg/100g dried sample) and 8% AGL (170mg/100g dried sample). These findings imply that addition of Ca on rice straw-based substrate can significantly improve yield, biological efficiency, and Ca content of the fruiting bodies.