The fermentation of hemicellulosic hydrolysate from Pinus taeda chips, using the fungal culture Rhizopus oryzae, was carried out to produce L-(+)-lactic acid and to optimize and enhance the biological conversion of reducing sugar into L-(+)-lactic acid using the experimental design to evaluate the culture conditions. The first factorial design based on surface response with five factors (agitation level, substrate concentration, CaCO3 concentration, C/N and C/P ratios) at low levels and one medium point was performed to optimize culture conditions. The second study tested two factors (substrate concentration and C/N ratio) at three levels. The statistical analysis of the data obtained from the factorial study showed that a C/N ratio of 35 and substrate concentration of 90 g/litre were the best conditions to produce L-(+)-lactic acid with R. oryzae on P. taeda hydrolysate, but in this case the statistical projection was not correct and the real optimized conditions were C/N ratio of 55 and substrate concentration of 75 g/litre of reducing sugar.