Fungal infection is alarming phenomenon now a day because of increasing number of patients. Lactophenol Cotton Blue is widely used for staining, but there is a necessity to develop an alternate equipotent stain which can replace it due to its high level of tumorigenic and hazardous nature. iodine glycerol is better alternative for it. Iodine glycerol is safe, eco-friendly, good visual clarity, with better staining properties. Hence, we got here various clinical sampleswhichwere analysed simultaneously using Lacto-phenol Cotton Blue and Iodine Glycerol by using different techniques like teasing technique, slide culture technique and adhesive tape technique. Parameters like degree of transparency, visual clarity, resolution, contrast, staining characteristics like uniformity, formation of artefacts were analysed for better demonstration of fungal morphology. Iodine-Glycerol is a better alternative to Lacto-phenol Cotton Blue for the demonstration of fungal morphology in the clinical microbiological laboratories. It is eco-friendly, noncarcinogenic and much potent staining reagent. It is necessary to carry further research as there are no specific guidelines regarding the preparation of the Iodine-Glycerol staining reagent. Keywords: LPCB, Iodine Glycerol; Lugol’s Iodine; Fungal Morphology; Transparency; Staining Characters.