Chip calorimetry is introduced as a new monitoring tool that provides real-time information about the physiological state of biofilms. Its potential for use for the study of the effects of antibiotics and other biocides was tested. Established Pseudomonas putida biofilms were exposed to substances known to cause toxicity by different mechanisms and to provoke different responses of defense and resistance. The effects of these compounds on heat production rates were monitored and compared with the effects of these compounds on the numbers of CFU and intracellular ATP contents. The real-time monitoring potential of chip calorimetry was successfully demonstrated by using as examples the fast-acting poisons formaldehyde and 2,4-dinitrophenol (DNP). A dosage of antibiotics initially increased the heat production rate. This was discussed as being the effect of energy-dependent resistance mechanisms (e.g., export and/or transformation of the antibiotic). The subsequent reduction in the heat production rate was attributed to the loss of activity and the death of the biofilm bacteria. The shapes of the death curves were in agreement with the assumed variation in the levels of exposure of cells within the multilayer biofilms. The new monitoring tool provides fast, quantitative, and mechanistic insights into the acute and chronic effects of a compound on biofilm activity while requiring only minute quantities of the biocide.