Temperature change over the length of heat exchangers might be an important factor affecting biofouling. This research aimed at assessing the impact of temperature on biofilm accumulation and composition with respect to bacterial community and extracellular polymeric substances. Two identical laboratory-scale plate heat exchanger modules were developed and tested. Tap water supplemented with nutrients was fed to the two modules to enhance biofilm formation. One “reference” module was kept at 20.0 ± 1.4 °C and one “heated” module was operated with a counter-flow hot water stream resulting in a bulk water gradient from 20 to 27 °C. Biofilms were grown during 40 days, sampled, and characterized using 16S rRNA gene amplicon sequencing, EPS extraction, FTIR, protein and polysaccharide quantifications. The experiments were performed in consecutive triplicate. Monitoring of heat transfer resistance in the heated module displayed a replicable biofilm growth profile. The module was shown suitable to study the impact of temperature on biofouling formation. Biofilm analyses revealed: (i) comparable amounts of biofilms and EPS yield in the reference and heated modules, (ii) a significantly different protein to polysaccharide ratio in the EPS of the reference (5.4 ± 1.0%) and heated modules (7.8 ± 2.1%), caused by a relatively lower extracellular sugar production at elevated temperatures, and (iii) a strong shift in bacterial community composition with increasing temperature. The outcomes of the study, therefore, suggest that heat induces a change in biofilm bacterial community members and EPS composition, which should be taken into consideration when investigating heat exchanger biofouling and cleaning strategies. Research potential and optimization of the heat exchanger modules are discussed.