The biotransformation potential of three phytosterols (campesterol, stigmasterol and β-sitosterol) under denitrifying, sulfate-reducing and fermentative/methanogenic conditions was assessed. Using a group contribution method, the standard Gibbs free energy of phytosterols was calculated and used to perform theoretical energetic calculations. The oxidation of phytosterols under aerobic, nitrate-reducing, sulfate-reducing and methanogenic conditions was determined to be energetically feasible. However, using semi-continuously fed cultures maintained at 20–22 °C over 16 weekly feeding cycles (112 days; retention time, 21 days), phytosterol removal was observed under nitrate-reducing and sulfate-reducing conditions, but not under fermentative/methanogenic conditions. Under sulfate-reducing conditions, stigmast-4-en-3-one was identified as an intermediate of phytosterol biotransformation, a reaction more likely carried out by dehydrogenases/isomerases, previously reported to act on cholesterol under both oxic and anoxic (denitrifying) conditions. Further study of the biotransformation of phytosterols under anoxic/anaerobic conditions is necessary to delineate the factors and conditions leading to enhanced phytosterol biodegradation and the development of effective biological treatment systems for the removal of phytosterols from pulp and paper wastewaters and other phytosterol-bearing waste streams.