Surface roughness is one of the key criteria for assessing surface condition across various industries. Even slight changes in topography can significantly affect the functional properties of components. Therefore, accurate roughness measurements require a comprehensive understanding of how measurement parameters influence the obtained results. This study analyses the impact of selected parameters of a digital optical microscope, as well as the characteristics of the light beam, on the accuracy of surface roughness reproduction compared to reference values. The investigation included the assessment of factors such as the direction of illumination, its intensity, depth composition step size, microscope magnification, and the resolution of the optical system. The main objective was to determine which of these parameters most significantly contribute to measurement distortions and to identify optimal settings that enable reliable and repeatable measurements. The presented analyses may constitute a valuable contribution to the development of metrological procedures, improving the reliability of surface roughness measurements in both industrial and research practices. They also facilitate better adaptation of microscope settings to the specific features of the analysed surface, which is crucial for ensuring proper interpretation of results.