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In this study, a new technique for three‐dimensional imaging of biofilm within porous media using X‐ray computed microtomography is presented. Due to the similarity in X‐ray absorption coefficients for the porous media (plastic), biofilm and aqueous phase, an X‐ray contrast agent is required to image biofilm within the experimental matrix using X‐ray computed tomography. The presented technique utilizes a medical suspension of barium sulphate to differentiate between the aqueous phase and the biofilm. Potassium iodide is added to the suspension to aid in delineation between the biofilm and the experimental porous medium. The iodide readily diffuses into the biofilm while the barium sulphate suspension remains in the aqueous phase. This allows for effective differentiation of the three phases within the experimental systems utilized in this study. The behaviour of the two contrast agents, in particular of the …