This in vitro study investigated how varying magnifications (5×, 10×, 20×, and 50×) using a confocal laser scanning microscope (CLSM) influence the measured surface roughness parameters, Ra/Sa and Rz/Sz, of various materials with two surface treatments. Cylindrical specimens (d ≈ 8 mm, h ≈ 3 mm, n = 10) from titanium, zirconia, glass-ceramic, denture base material, and composite underwent diamond treatment (80 μm; wet) and polishing (#4000; wet; Tegramin-25, Struers, G). The surface roughness parameters (Ra/Sa, Rz/Sz) were measured with a CLSM (VK-100, Keyence, J) at 5×, 10×, 20×, and 50× magnifications. Line roughness (Ra/Rz) was measured along a 1000 μm distance in three parallel lines, while area roughness (Sa/Sz) was evaluated over a 2500 μm × 1900 μm area. The statistical analysis included ANOVA, the Bonferroni post hoc test, and Pearson correlation (SPSS 29, IBM, USA; α = 0.05). Ra/Sa and Rz/Sz showed significant differences (p ≤ 0.001, ANOVA) across magnifications, with values decreasing as magnification increased, highest at 5× and lowest at 50×. Titanium, zirconia, and glass-ceramic showed significant measured roughness values from 5× to 50×. Denture base material and composite had lower measured roughness values, especially after polishing. Line and area roughness varied significantly, indicating that magnification affects measured values. Standardizing magnifications is essential to ensure comparability between studies. A 50× magnification captures more detailed profile information while masking larger defects.