Teachers’ well-being at work is an important indicator of their mental health. Strengths use has been identified as a significant predictor of enhanced well-being at work. However, there is a scarcity of studies that have examined the connection between teachers’ strengths use and well-being at work; thus, its underlying psychological mechanism is unclear. Therefore, this study aimed to explore the association between teachers’ strengths and well-being at work together with the mediating role of basic need satisfaction. A total of 374 university teachers completed a series of questionnaires on strengths use, basic need satisfaction, hedonic well-being, and eudaimonic well-being. The results showed that there were positive correlations between strength use and both types of well-being at work. Moreover, basic need satisfaction mediated the association between strength use and two types of well-being at work. The findings suggest that institutions should prioritize enhancing teachers’ ability to utilize their strengths and foster an environment conducive to such practices, thereby improving their workplace well-being.