Biofouling causes significant losses in efficiency in heat exchangers recovering waste heat from treated sewage. The influence of the temperature field on biofouling was investigated using a flat plate heat exchanger which simulated the channels in a plate and frame unit. The test surface was a 316 stainless steel plate, and a solution of Bacillus sp. and Aeromonas sp. was used as a model process liquid. The test cell was operated under co-current, counter-current, and constant wall temperature configurations, which gave different temperature distributions. Biofouling was monitored via changes in heat transfer and biofilm thickness. The effect of uniform temperature on biofouling formation was similar to the effect of uniform temperature on planktonic growth of the organisms. Further results showed that the temperature field, and particularly the wall temperature, influenced the rate of biofouling strongly. The importance of wall temperature suggests that fouling could be mitigated by using different configurations in summer and winter.