Natural rubber (NR) powder wastes contribute to the pollution of the environment and pose a risk to human health. Therefore, Escherichia coli AY1 and Aspergillus oryzae were used to degrade NR in the present investigation. The biodegradation was further confirmed using E. coli AY1 and A. oryzae’s ability to create biofilm, which grew on the surface of the NR. Additionally, the biodegraded NR was examined by scanning electron microscopy (SEM), attenuated total reflection−Fourier transform infrared (ATR−FTIR) spectroscopy, and gas chromatography−mass spectrometry (GC−MS). The highest weight loss (69%) of NR was detected (p < 0.05) after 210 d of incubation with the mixed microbial culture (E. coli AY1 + A. oryzae). In the SEM, the surface of the control treatment appeared uniform and normal, whereas the surface of the microbial treatment displayed an irregular shape, with apparent particle deformation and surface erosion. After biodegradation by E. coli AY1 and A. oryzae, the particle size range of the untreated NR dropped from (5.367−9.623 µm) to (2.55−6.549 µm). After treating NR with E. coli AY1 and A. oryzae, new bands appeared in the ATR−FTIR technique; others shifted down in the range of 3910−450 cm−1, suggesting the existence of active groups belonging to alcohol, secondary amine, aromatic amine, conjugated anhydride, aldehyde, alkene, and halo compounds. On the other hand, the GC−MS profile reports a significant decline (p < 0.05) in the amount of hydrocarbons while simultaneously reporting a significant increase (p < 0.05) in the proportion of oxygenated, sulfurous, and nitrogenous compounds. These active groups are attributed to the antioxidant and antibacterial properties of biodegraded NR by a mixture of E. coli AY1 and A. oryzae, which rose 9-fold (p < 0.05) compared to untreated NR. Through the use of this research, we will be able to transform NR waste into a valuable product that possesses both antioxidant and antibacterial properties.