Geobacillus sp. D413 and Geobacillus toebii E134 are aerobic, non-pathogenic, endospore-forming, obligately thermophilic bacilli Grampositive thermophilic bacilli can produce heat-resistant spores. The bacteria are indicator organisms for assessing the manufacturing process's hygiene and are capable of forming biofilms on surfaces used in industrial sectors. The present study aimed to determine the biofilm-forming properties of Geobacillus isolates and how to eliminate this formation with sanitation agents. According to the results, extracellular DNA (eDNA) was interestingly not affected by the DNase I, RNase A, and proteinase K. However, the genomic DNA (gDNA) was degraded by only DNase I. It seemed that the eDNA had resistance to DNase I when purified. It is considered that the enzymes could not reach the target eDNA. Moreover, the eDNA resistance may result from the conserved folded structure of eDNA after purification. Another assumption is that the eDNA might be protected by other extracellular polymeric substances (EPS) and/or extracellular membrane vesicles (EVs) structures. On the contrary, DNase I reduced unpurified eDNA (mature biofihns). Biofilm formation on surfaces used in industrial areas was investigated in this work: the D413 and E134 isolates adhered to all surfaces. Various sanitation agents could control biofilms of Geobacillus isolates. The best results were provided by nisin for D413 (80%) and a-amylase for E134 (98%). This paper suggests that sanitation agents could be a solution to control biofilm structures of thermophilic bacilli.