In situ remediation is usually restricted by temperature, lack of substrate for reductive dechlorination (anaerobic respiration), the presence of dehalogenating microorganisms, and specific bedrock conditions. In this work, trichloroethene (TCE) degradation was studied by a number of methods, from physical–chemical analyses to molecular biological tools. The abundance changes in dechlorinating bacteria were monitored using real-time PCR. The functional genes vcrA and bvcA as well as the 16S rRNA specific for representatives of genera Dehalococcoides, Dehalobacter, and Desulfitobacterium were monitored. Furthermore, the sulfate-reducing bacteria and denitrifying bacteria were observed by amplifying the functional genes apsA and nirK. The elevated temperature and the substrate (whey) addition significantly affected TCE dechlorination. The chlorine index decreased after nine weeks from 2.5 to 0.1 at 22 °C, to 1.1 at 17 °C and 1.7 at 12 °C and complete dechlorination was achieved at 22 °C with whey addition. The achieved results of this work show the feasibility and effectiveness of biological dechlorination of TCE enhanced with elevated temperature and whey addition.