Recent research points to decreasing testosterone (T) levels as well as decreasing relationship quality during the transition to fatherhood, and it has been suggested that T reflects and affects motivation and behavior with respect to mating or paternal effort. Accordingly, we hypothesized that decreases in T are associated with decreasing relationship quality in new fathers. Thirty-seven fathers and 38 men in committed romantic relationships without children (controls) were recruited. All subjects participated actively by collecting saliva samples for T assessment three times a day on two assessment days, four weeks prior to birth (day 1) and eight weeks after birth (day 2) for fathers, and three months after the first assessment for controls and by filling out questionnaires on relationship quality. Results revealed significantly lower T levels (AUCg-T) in fathers than in controls at day 2 and significant decreases in relationship quality from day 1 to day 2 in fathers, but not in controls. In particular, the new fathers reported tenderness in their relationship to have significantly decreased from pre to post birth in comparison to the controls. These results were partially moderated by T levels at day 1. We interpret our results as being in line with the "challenge hypothesis" in humans, according to which T levels are positively associated with mating effort and negatively related to paternal activities.