A novel thermophilic Bacillus sp. capable of producing lipase was locally isolated. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Bacillus thermoleovorans. Plackett-Burman experimental design was used to evaluate cultural conditions affecting lipase production process. Fifteen variables and four dummy variables were examined in the experimental design. Tween 80, temperature, olive oil, aeration, beef extract and inoculum age were found to be the highest positive significant variables affecting lipase activity, whereas pH and calcium chloride were the highest negative significant variables. Moreover, Tween 80, temperature and olive oil positively affected lipase specific activity. On the other hand, gum arabic, inoculum size and calcium chloride had the highest negative effect on lipase specific activity. This study would improve the further optimization steps on the bioprocess development track.