Aim: The aim of this in vitro study was to evaluate the effect of glass and nylon fiber reinforcement on surface topography of polymethyl methacrylate (PMMA) denture resin after polishing. Materials and methods: Stainless steel dies were used for preparation of specimens and were divided into three groups of 30 specimens each. Group A: Control group, Group B: PMMA reinforced with glass fibers, Group C: PMMA reinforced with nylon fibers. All specimens were finished and polished by a single operator to eliminate any inadvertent bias and ensure a constant pressure when polishing. The surface roughness of all the acrylic samples was measured with the help of profilometer (SURFCOM 130A). Results: Glass-reinforced PMMA showed higher mean surface roughness (0.16817 µm) as compared to unreinforced PMMA (0.10203 µm). Nylon-reinforced PMMA showed lower mean surface roughness (Ra) of 0.09177 µm as compared to unreinforced PMMM (0.10203 µm). Glass-reinforced PMMA showed a higher mean surface roughness (Ra) of 0.16817 µm as compared to nylon PMMA (0.09177 µm). Conclusion: Reinforcement with glass and nylon fibers affects the surface roughness of the PMMA resin, i.e., glass fibers increase the surface roughness of PMMA resin, whereas nylon fibers slightly decrease the surface roughness of PMMA resin. Hence, the use of nylon fiber may be justified to obtain a denture with increased fracture resistance, acceptable esthetic properties, and better denture hygiene and plaque accumulation. Clinical significance: Two important parameters are essential for ensuring the durability of a restorative material, which include surface roughness and color stability. Surface roughness may be the major cause of discomfort for the patient and it may be associated with complications by promoting the deposition of biofilm and microbial growth. The use of nylon fiber increases fracture resistance and provides acceptable esthetic properties and better denture hygiene and plaque accumulation.