Low-density polyethylene (LDPE) is a widely used thermoplastic polymer in the production of plastic bags and pipes due to its flexibility, chemical resistance, and low cost. However, its recalcitrance to degradation contributes significantly to global plastic pollution, underscoring the need for strategies to enhance its breakdown. This study assessed the physicochemical alterations in LDPE beads after 30 days of exposure to native Colombian fungal strains of Fusarium oxysporum (FOCIC01), under conditions where LDPE served as the sole carbon source. The experimental setup included mineral salt medium inoculated with the FOCIC01 strain. Following incubation, LDPE beads were separated from fungal biomass via sequential washing with 1% SDS, rinsing with 70% ethanol and sterile distilled water, followed by sonication to ensure thorough removal of biofilm and surface residues prior to analysis. Structural and chemical changes in the polymer were analysed through Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF). SEM revealed pronounced surface erosion, fissures, and pitting in LDPE beads exposed to the fungal strain, in contrast with the smooth, intact control beads. FTIR analysis showed the appearance of new absorption bands corresponding to hydroxyl (–OH) and carboxyl (–COOH) groups, indicating oxidative modification of the polymer structure. These spectral changes suggest the occurrence of oxidative degradation processes, likely facilitated by fungal enzymatic activity. MALDI-TOF mass spectrometry, performed on the supernatants of the degradation assays, revealed fragmentation patterns indicative of partial depolymerisation, with signals consistent with low-molecular-weight polyethylene oligomers. The integration of these complementary analytical approaches demonstrates the potential of native F. oxysporum to initiate degradation of LDPE through extracellular mechanisms. This work provides new insights into polymer–fungus interactions and supports the development of fungal-based biodegradation strategies for synthetic plastic waste.