Studies in mammals have implicated the neuropeptide oxytocin (OT) in processes of bond formation and stress modulation, yet the involvement of OT in human bonding throughout life remains poorly understood. We assessed OT in the plasma, saliva, and urine of 112 mothers and fathers interacting with their 4–6-month-old infants. Parent–infant interactions were micro-coded for parent and child’s social behaviors and for the temporal coordination of their socio-affective cues. Parents were interviewed regarding their attachment to the infant and reported on bonding to own parents, romantic attachment, and parenting stress. Results indicated that OT in plasma (pOT) and saliva (sOT) were inter-related and were unrelated to OT in urine (uOT). pOT and sOT in mothers and fathers were associated with parent and child’s social engagement, affect synchrony, and positive communicative sequences between parent and child. uOT was related to moments of interactive stress among mothers only, indexed by the co-occurrence of infant negative engagement and mother re-engagement attempts. pOT and sOT were associated with mothers’ and fathers’ attachment relationships throughout life: to own parents, partner, and infant, whereas uOT correlated with relationship anxiety and parenting stress among mothers only. Similar to other mammals, OT is involved in human attachment and contingent parenting. The dual role of OT in stress and affiliation underscores its complex involvement in processes of social bonding throughout life.