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The use of an ultrasonic apparatus (40 kHz) for the non‐destructive, rapid and reproducible removal of biofilm from standard materials (stainless steel and polypropylene) in a dairy factory was investigated. The application of ultrasound with the tested conditions (10 s and 40 kHz) was found not to be detrimental for standard ATP (concentration ranging between 5 × 10⁻⁹ and 10⁻⁵ mol l⁻¹) and for prokaryotic cells, including both rods and coccoid‐shaped bacteria (Escherichia coli and Staphylococcus aureus). It allowed the use of the ATP bioluminescence measurement for quantifying the biofilm removal. The repeatability of industrial milk removal was determined on fouled stainless steel and polypropylene sheets. The variability of the results with the sonication method was constant, ±24% (coefficient of variation) for both surfaces, and was variable with the swabbing method, ±42% for the stainless steel sheet …