Environmental pollution from packaging, has led to a need for sustainable alternatives. This study investigates the biodegradation of polylactic acid (PLA) by Amycolatopsis orientalis and Amycolatopsis thailandensis after thermal and thermal-alkaline pretreatments. The biodegradation was assessed based on weight loss, CO2 evolution, carbon balance analysis and scanning electron microscopy (SEM). The analysis showed that pretreatment at 37 °C for 8 h provided effective enhancement of the biodegradation performance. Combining thermal pretreatment with alkaline conditions led to chemical degradation of PLA, but is less suitable as a pretreatment for biodegradation. It was also demonstrated that the mineralization rate over a two-week period was higher following thermal than thermal-alkaline pretreatment. SEM confirmed improved biodegradation as illustrated by increased surface roughness. These findings suggest that thermal pretreatment at 37 °C for 8 h is the most effective strategy for enhancing PLA biodegradation by Amycolatopsis spp., promoting a sustainable approach to plastic waste management.