Office layout plays a significant role in shaping indoor environmental quality (IEQ) and its subjective perception by occupants. This paper aims to compare the perception of selected IEQ factors in office buildings with standard and open-space layouts. The evaluation was conducted through a questionnaire survey focusing on thermal comfort, indoor air quality (IAQ), lighting, and acoustic comfort, as well as the perceived impact of these factors on occupants’ comfort and work productivity. In total, 46 respondents participated in the survey. Descriptive statistics and the non-parametric Mann–Whitney U test were used to analyze the collected data. Statistically significant differences were identified in the subjective perception of acoustic comfort (noise) and indoor air quality (stuffiness) between the compared office layouts, whereas no significant differences were found for thermal and lighting comfort. Overall, the findings contribute to a better understanding of how office layout influences perceived IEQ and may support the sustainable and user-oriented design of office spaces.