Polyamide is a thermoplastic polymer widely used for several applications, including cables in offshore oil and gas operations. Due to its growing annual production worldwide, this poorly biodegradable material has been a source of pollution. Given this scenario, the need has arisen to develop environmentally friendly techniques to degrade this waste, and biotechnology has emerged as a possible solution to mitigate this problem. This study aimed to investigate the potential of Yarrowia lipolytica to biodepolymerize polyamide fibers (PAF). Microbial cultures were grown in shaken flasks containing different concentrations of PAF (0.5 and 2 g·L−1) and in a bioreactor with and without pH adjustment. PAF mass loss was up to 16.8%, achieved after 96 h of cultivation in a bioreactor without pH adjustment. Additionally, NMR analyses revealed that the amorphous regions of PAF, which are more susceptible to depolymerization, were reduced by 6% during cultivation. These preliminary results indicate the biotechnological potential of Y. lipolytica to depolymerize PAF.