A synergistic interaction between reduced graphene oxide (rGO) and a biodegradable natural polymer, sodium alginate, was developed to create unique microspheres with protruding spiky features at the surface (spiky microspheres) that act as a super encapsulation and sustained release system for the highly effective antibiotic cefotaxime. Three forms of microspheres, namely alginate (Alg), alginate-cefotaxime (Alg-CTX), and alginate-cefotaxime-reduced graphene (Alg-CTX-rGO) composites, were prepared using calcium chloride as a cross-linking agent. The microspheres were characterized using field emission scanning electron microscopy (FESEM), Fourier-transform infrared (FT-IR) spectroscopy, and X-ray diffraction to investigate their pores, roughness, surface morphology, functional groups, phase formation, purity, and structural properties. The membrane diffusion method was employed to determine the release profile of Cefotaxime from the fabricated microspheres. The antibacterial activities of CTX solution, Alg microspheres, Alg-CTX microspheres, and Alg-CTX-rGO microspheres were investigated against gram-negative bacteria (Escherichia coli) using the agar diffusion method on Muller–Hinton agar. The prepared samples exhibited excellent results, suggesting their potential for enhanced antibiotic delivery. The results demonstrated the potential of the microsphere 2D rGO/alginate matrix for enhancing cefotaxime delivery with an unusual, prolonged release profile.