To achieve organizational goals, one of the most important tools in complex and variable environments is strategic flexibility. Strategic flexibility refers to the organization's ability to respond to environmental changes. The present study seeks to investigate the effect of organizational ambidexterity on performance with the mediating role of strategic flexibility in small and medium-sized companies in Mashhad. For this purpose, by adopting a quantitative and survey research method, the questionnaire of Kaftzopoulos et al. (2023) was used. Validity was confirmed by factor analysis, content validity, and reliability by Cronbach's alpha. The statistical population included managers of small and medium-sized companies, the number of whom was unknown and non-random sampling method was available. The thumb rule method was used to determine the sample size. The number of necessary samples is equivalent to 5 to 15 times the number of questionnaire items, and the minimum number of samples required for data collection was 100 questionnaires. 120 acceptable questionnaires were used for data analysis and data analysis was performed in the PLS format. The results showed that ambidexterity had a significant effect on the variables of strategic flexibility and business performance, both in terms of exploration and exploitation. Business flexibility was also found to have an effect on business performance. The mediating effect of strategic flexibility on the relationship between ambidexterity and performance was also confirmed. The overall results indicate that ambidexterity, through the ability of the organization to respond to environmental requirements and changes, is a reliable approach to improving the performance of organizations.