Interest on biofuel production from biomass and biofiber has gain great attention globally because these materials are abundant, inexpensive, renewable, and sustainable. Generally, the conversion of biomass and biofiber to biofuel involves several processes including biomass production, pretreatment, hydrolysis, and fermentation. Selecting the most efficient pretreatment is crucial to ensure the success of biofuel production since pretreatment has been reported to contribute substantial portion on the production cost. The main goal of the pretreatment is to enhance digestibility of the biomass and biofiber, and to increase sugar production prior to fermentation process. To date, several pretreatment methods have been introduced to pretreat biomass and biofiber including irradiation. This book chapter reviews and discusses different leading irradiation pretreatment technologies along with their mechanism involved during pretreatment of various tropical biomass and biofiber. This chapter also reviews the effect of irradiation pretreatment on the biomass and biofiber component, which could assist the enzymatic saccharification process.