Microplastics (MPs) pose potential public health challenges because of their widespread occurrences in all environmental compartments. While most studies have focused on the occurrence fate of microplastics in wastewater treatment systems, the biodegradation of microplastics in wastewater is generally little understood. Therefore, we used two Gram-positive and thermophilic bacteria, called strain ST3 and ST6, which were identified by morphological, biochemical, physiological, and molecular analyses, to assess the growth and biodegradation potential of two different sized (50 and 150 m) polyethylene particles. The degradation was monitored based on structural and surface morphological changes. According to 16S rRNA analyses, ST3 and ST6 were identified as Anoxybacillus flavithermus ST3 and Anoxybacillus sp. ST6, respectively. The occurrence of cracks, holes, and dimensional changes was detected by scanning electron microscopy. Moreover, critical characteristic absorption band formation and modifications were determined by Fourier transform infrared spectroscopy. In addition to these, it was found that Anoxybacillus flavithermus ST3 and Anoxybacillus sp. ST6 produced high level of alpha-Amylase. These results showed that thermophilic bacteria are capable of the biodegradation of microplastics and production of alpha-Amylase.