This study aims to find effective factors for the production of organic solvent-stable lipase from Acinetobacter baylyi that has been isolated from marine sludge in Angsila, Thailand. A study of physical parameters for lipase production by this strain revealed an optimum condition to be 25°C, pH 5.75 at 150 rpm. Maximum lipase activity and biomass contents were achieved after cultivation of the strain at the optimum condition for 15 h. Lipase production could be enhanced to nearly 2.5-fold by using glucose and ammonium sulfate at a concentration of 0.8% (w/v) and 0.4% (w/v) in the culture medium, respectively. Moreover, a 24- fold higher activity was observed with a combination of glucose and ammonium sulfate. When 0.8% (w/v) tryptone was included in the growth medium, lipolytic activity in the strain could be increased ~ 8.5-fold after 24 h of growth. The addition of either 5mM alanine or 0.1% (w/v) agar to minimal medium gave lipase production ~7-fold, approaching that obtained in the same medium. No significant lipase production was observed with the addition of hexadecane. Interestingly, the addition of 0.8% (v/v) of Tween 80 could enhance the enzyme production with 16,142- fold. A.baylyi lipase tolerated up to at least 75% (v/v) of short-chain alcohols, acetonitrile, heptane and decane and was also stable in the presence of 25% (v/v) DMSO, benzene, hexanes and hexadecane. This organic solvent-stable lipase could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvent.