A time-dependent, heat diffusion equation was used to predict the three-dimensional temperature distribution of Escherichia coli in liquid-suspension during irradiation by a high-power Nd:YAG laser. The model may be used to calculate the temperature rise and the transient 3-D temperature profile in the liquid suspension under arbitrary combinations of laser wavelength, pulse shape, pulse width, repetition rate, energy density, and for different concentrations of bacteria. The temperature profiles in the liquid, for a range of energy densities, were measured to validate the theoretical model. The experimental results were in good agreement with the theoretical ones. A temperature gradient was found in the sample in the radial and axial directions during laser irradiation. The model enables the parameters that affect the temperature distribution of the liquid suspension to be identified and optimized when designing laser sterilization systems.