The strikes took place in the village of Mayfadoun near Nabatiyeh as medical teams responded to emergency calls.
The first strike killed two paramedics from the Islamic Health Committee. A second team arriving to assist the wounded was then hit, injuring additional medics.
A third rescue team, including members of the Nabatiyeh Emergency Services and the Islamic Risala Scout Association, was also struck, killing two more rescue workers.
The Israeli military said it was "looking into" the incident and has previously accused Hezbollah of using ambulances for militant activity, without providing evidence.
Lebanon's Health Ministry condemned the attacks as a "blatant violation" of international law.
Aid groups warned that repeated strikes on emergency responders are eroding protections for medical teams, though they pledged to continue rescue operations.
The violence comes amid intensified fighting in southern Lebanon. Israel said it struck more than 200 Hezbollah targets in 24 hours, while Hezbollah reported rocket fire toward northern Israel.
Prime Minister Benjamin Netanyahu said he ordered an expansion of a buffer zone in southern Lebanon and reiterated that Israel is pursuing both military action and negotiations aimed at disarming Hezbollah and achieving "sustainable peace."
The conflict has worsened Lebanon's humanitarian crisis, with over 2,000 killed since the escalation, including at least 91 medical workers, and widespread displacement forcing many civilians into overcrowded areas like Tyre, now considered one of the last relatively safer locations. (AP)