The fire started around 7 a.m. at the house in an alley on Mac Thien Tich Street.
Nearly 10 people who rented rooms inside rushed out but Nguyen Thi Ngoc Mai, 16, and Nguyen Huyen Mi, 19, were stuck upstairs.
Three fire trucks were dispatched to the site along with 14 firefighters.
It took them around 20 minutes to kill off the fire.
The two-story house was built over 40 sq.m. and divided into 15 rooms to offer accommodation for people who come to HCMC to seek health checks at the Ho Chi Minh City Medical and Pharmaceutical University Hospital, the city's largest public facility for the purpose. These people often rent a room for one or two nights.
Nguyen Xuan Thanh, a District 5 official, said initial investigation suggested that the fire started from a mattress in one of the rooms.
Police are investigating further.