The nutritional value and yield of mushrooms depend on the substrate on which it is grown. This study sought to biofortify Pleurotus floridanus with calcium supplements and assess its effect on the yield and calcium levels. The experiment was set up in a 2 × 5 factorial and replicated thrice in a completely randomized design. Two calcium supplements, OML and OMW, were added to two growth media. The examination of total dry weight yield showed that calcium supplements OML and OMW in the sawdust medium containing wheatbran in the ratio 1:10 had a mean value of 4.37 g, which was significantly higher (P < 0.05) than that in the control (1.29 g). However, in the sawdust-only medium, there was no significant difference (p > 0.05) in the application of treatments. No significant difference (p > 0.05) was observed between the calcium types in both growth media. The mineral analysis showed that calcium levels were increased in harvested mushrooms with the addition of calcium OML and OMW to the growth media.