Bacillus pumilus D194a formed a strong biofilm on 96-well polystyrene microtiter plates and stainless-steel surfaces. Its optimum environmental factors for growth were determined as 50 degrees C, pH 6.0, 1.5% NaCl for 48 h, whereas, the optimum biofilm formation was measured at 50 degrees C, pH 8.0, 0% NaCl. The isolate was observed to have peritrichous flagella and it produced rigid pellicle. Extracellular polymeric substances (EPSs) of this strain were also determined to be; carbohydrate (33.5 mu g/mL), protein (790 mu g/mL) and extracellular DNA (eDNA) (18.5 kb). D194a cells formed biofilms on many abiotic surfaces, such as glass, polystyrene, stainless steel, polyvinyl chloride, polycarbonate, polypropylene, wherein polycarbonate (6.23 log/cm(2)) and polypropylene (6.14 log/cm(2)) were the generally preferred ones. Biofilm samples were treated with 15 different sanitation agents, including sodium metaperiodate (96.83%), nisin (93.82%), trichloroacetic acid (93.66%) and lysozyme (94.1%) for sanitation purposes. The elimination of eDNA in its 2 h-biofilm with DNase I was 98.48%, while this results was found as 96.06% for its 12 h-mature biofilm. In conclusion, this research demonstrated that B. pumilus D194a is a strong biofilm producer with a rigid structure harboring high protein and eDNA content, which cannot be easily destroyed by various chemical agents.