This empirical study aimed to analyse the influence of the personality traits of owner-managers in small and medium-sized enterprises (SMEs) on organisational ambidexterity (OA). Based on the existing literature, five hypotheses were formulated about the relationships between the Big Five personality traits and organisational ambidexterity. A second-order structural equation model was used with a sample of 224 Portuguese SMEs in the sector of information technology (IT), telecommunications, and audio-visual and IT consultancy. The results obtained suggest that the personality traits of extraversion, neuroticism (versus emotional stability) and conscientiousness have a significant influence on organisational ambidexterity. These results are consistent with the research and demonstrate that owner-managers’ personality traits influence organisational ambidexterity in SMEs. Theoretical and practical implications are explored.