The formation of biofilm on hydrocarbon in a porous medium under biostimulation conditions was investigated in order to a better understanding of microbial development during bioremediation of hydrocarbon‐contaminated soils. Pseudomonas alkanolytica ATCC 21034 was studied as it grows embedded in a biofilm on dodecane surface. First, an experimental protocol for determination of biofilm growth kinetics on hydrocarbon in a porous medium was proposed. Six identical columns packed with sand were run simultaneously. The growth kinetics was obtained by measuring the biofilm dry weight of each column at different times. The reproducibility of the experimental protocol was validated using statistical tests. Then biofilm growth kinetics was established in the porous medium for two specific surfaces of dodecane (large or small droplets). A linear growth was observed due to transport limitation of both dodecane and dissolved oxygen in the biofilm. So the degradation of dodecane was uncompleted. Moreover, difficulty to bring dissolved oxygen in the deeper part of the porous medium was pointed out. All these phenomena could explain some failures encountered during biostimulation processes. © 2007 American Institute of Chemical Engineers Environ Prog, 2007