This study aimed to investigate the effect of organizational culture on turnover intentions and whether this relationship is mediated by affective commitment. For this purpose, the following hypotheses were formulated: (1) organizational culture (support culture, innovation culture, goal culture, rule culture) has a negative and significant effect on turnover intentions; (2) organizational culture (support culture, innovation culture, goal culture, rule culture) has a positive and significant effect on affective commitment; (3) affective commitment has a negative and significant effect on turnover intentions; (4) affective commitment mediates the relationship between organizational culture (support culture, innovation culture, goal culture, rule culture) and turnover intentions. A total of 345 individuals working in Portuguese organizations participated in this study. The results indicate that all dimensions of organizational culture have a negative and significant effect on turnover intentions and a positive and significant effect on affective commitment, with support culture having the strongest effect on both affective commitment and turnover intentions. Affective commitment has a negative and significant effect on turnover intentions. Affective commitment partially mediates the relationship between three dimensions of organizational culture (support culture, goal culture, and rule culture) and turnover intentions. The mediation effect of affective commitment in the relationship between innovation culture and turnover intentions is total. These results indicate that organizations should foster a strong organizational culture, especially support culture, so that their employees feel more affectively committed to the organization and reduce their turnover intentions.