This study deals with the possible degradation of toluene and acetic acid when subjected to cell-free enzyme system from the toluene degrading bacteria Pseudomonas putida and acetic acid degrading bacteria Cupriavidus necator. P. putida produces toluene dioxygenase only under the existence of toluene in culture medium and toluene is degraded to cis-toluene dihydrodiol by this enzyme. C. necator produces acetyl coenzyme A synthetase-1 and converts acetic acid to acetyl CoA in order to synthesize ATP to need for growth or PHA which is biodegradable polymer. In case of toluene degradation, the experiment was conducted before and after production of toluene dioxygenase as this enzyme, produced by P. putida, is an inducible enzyme. Toluene was detected using gas chromatography (GC). Similar amount of toluene was found in control group and before production of toluene dioxygenase (experimental group 1). However, reduction in toluene was detected after the production of toluene dioxygenase (experimental group 2). Acetic acid was detected through application of gas chromatography-mass spectrometer (GC-MS). The results showed the acetic acid peak was not detected in the experimental group to apply cell-free enzyme system. These results show that the cell-free enzyme system obtained from P. putida and C. necator retained the ability to degrade toluene and acetic acid. However, P. putida needs to produce the inducible enzyme before preparation of the cell-free enzyme system.