A thermophilic lipase-producing bacterium (Anoxybacillus sp. HBB16) was analyzed using 16S rRNA. The maximum growth rate and intracellular lipase production occurred at 50 °C and pH 6.5. Among the various nitrogen and carbon sources tested, meat extract, olive oil and olive mill wastewater (OMW) were the best sources for lipase production. Enzyme production increased when the strain HBB16 was grown at a 180 rpm shaking speed. The maximum activity of the lipase occurred at 55 °C and pH 9.5. The presence of phenylmethylsulfonyl fluoride (PMSF), N-cyclohexyl-N′-(2-morpholinoethyl) carbodiimidemetho-p-toluenesulfonate (CMC), N-bromosuccinimide (NBS) and sodium dodecyl sulfate (SDS) inhibited enzyme activity. Bivalent metal ions caused a significant inhibition in enzyme activity, whereas univalent metal ions displayed no negative effects.