Water Hyacinth (Eichhornia crassipes) is a weedy lignocellulosic material which represents the best substrate for the production of bioethanol in tropical countries due to its more availability and more biomass yield. For the fermentation and bioethanol production from water hyacinth, the different bacterial and fungal inoculants were used. Water hyacinth was obtained from ukkadam-valankulam lake, Coimbatore. These leaves were washed properly to remove soil and contaminants and dried for 3 hours at 120°C. It was grained into a fine powder and sieved using 1.5µm in diameter nylon sieve. The sieved material was cultured using different microbial culture such as Aspergillus oryzae, Aspergillus niger and Saccharomyces cerevisiae. After 21 days incubation, the culture was distilled using rotary vacuum evaporator and the produced bioethanol was analyzed by Fourier-transform infrared spectroscopy (FTIR) technique. The previous report showed, less yield only obtained using water hyacinth, potato peels, cassava peel and millet husks but this study produced highest yield using water hyacinth. This study indicated that water hyacinth is mostly available aquatic plant for the production of ethanol. The aim of current work is to understand the usage of water hyacinth as main substrate for bioethanol production using fungal inoculum. The microbial inoculants used for current study reported more potential for the production of bioethanol. This current work gives more economical value to water  hyacinths and it can be cleared from all the waterways.