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The harvesting and hauling operations of bioenergy feedstock is an important area in biofuel production. Production costs can be minimized by maintaining optimal machinery units for these operations. The objective of this study is to design an optimal harvesting unit for bioenergy refinery and estimate harvesting and hauling costs of energy cane. A biorefinery with the annual capacity of processing twenty-five million imp. gallons of ethanol were considered. Given the efficiency of harvesting, a two-row soldier system was considered. Considering the year-round supply of energy cane to the refinery, the optimal machinery unit was designed, and the combined operation costs were derived. The average estimated ownership, repair, labor and fuel and lubricant costs of biomass harvest unit were calculated to be $0.50, $0.54, $1.78 and $1.51/mt, respectively. The costs distribution generated showed harvesting and hauling costs could range between $5.47–$9.23/mt of energy cane. The methodology and the research output will provide guidelines for investors in designing harvesting and hauling units and estimating costs for different scales of operation.