Polyurethanes (PUs) are characterized by different physicochemical and mechanical properties that enable their utilization in a wide range of applications. Recently, biobased polyurethanes have attracted great interest as an ecofriendly alternative to traditional polyurethanes. These biobased macromolecules display unique properties, such as renewability, low toxicity and abiotic or biotic degradability. This research investigated the biodegradation of biobased polyurethane films by different fungi, namely, Aspergillus niger (An), Penicillium sp. (Pen) and Trichoderma hazardium (Th). The biodegradation tests were performed under static conditions and in liquid media. The biodegraded polyurethane films were characterized using ATR-FTIR, thermal analysis and scanning electron microscopy (SEM). The chemical structure of the biodegraded samples demonstrated obvious evidence for degradation through the disappearance or shifting of absorption bands according to the fungal strain used. Thermal degradation investigations confirmed the effect of fungi on the hard segment of the urethane bonds in the polyurethans films, leading to their decomposition at lower temperatures than in the case of the untreated films. Additionally, surface imaging of the treated films revealed significant changes after exposure to metabolic reactions of the fungi used. The results confirmed that the presence of amino groups in the PU structure enhances polymer disintegration. Moreover, the effect of Trichoderma hazardium was more pronounced than that of the other strains.