The outbreak in the West African country is already thought to be the fourth largest on record but could escalate after reaching a densely populated city.
The country's Health Ministry confirmed several cases in the port of Bata, while the US embassy in the country gave the number of cases in the city as four.
US officials advised against all but essential travel to the mainland of the country of 1.5 million and restricted transport for its own staff.
Marburg virus belongs to the same filovirus family as the better-known Ebola and in the worst previous outbreaks has been known to kill nearly 90 per cent of those infected.
The outbreak was first confirmed last month but officials last week acknowledged it was larger and more widespread than initially thought.
There have been nine laboratory-confirmed cases, of which seven are dead and two are undergoing treatment. There have been another 20 suspected cases, all of whom are now dead.
Cases have been recorded in three provinces right across the country, over a range of about 145km, suggesting doctors had been unable to keep track of infected people or stop them from passing on the virus.
Monitoring has been hampered by the outbreak arising in a remote rural corner of Kie-Ntem province.
People ignoring quarantine restrictions there had been responsible for spreading the infection to Bata, the Health Ministry said.
Teresa Lambe, professor of vaccinology and immunology at the Pandemic Sciences Institute, the University of Oxford, said: “There are currently no licensed vaccines or treatments for Marburg virus disease, so we rely yet again on contact-tracing to prevent even more casualties.
“We need to take pandemic preparedness more seriously and invest in vaccines, treatments and clinical trials to prevent these dangerous viruses from spreading.”
The arrival of the virus in Bata, a major cargo port from which flights to neighbouring countries are available, will heighten fears that the disease may spread beyond Equatorial Guinea's borders.
Neighbouring Gabon and Cameroon are already on high alert.
Health officials in Cameroon said they had tested 15 suspected cases between March 6 and 19 but said none were confirmed.
Egyptian fruit bats are considered the natural animal host of the virus, though it has also been found in monkeys.
When the virus spreads to humans, it then spreads from person to person via bodily fluids such as blood and mucus, as well as through contact with bedding and clothing.
Previous outbreaks have resulted in health workers and those preparing bodies for burial to be particularly at risk.
Symptoms begin with high fever, severe headache and malaise. Many patients start to haemorrhage after five to seven days, bleeding from their bodily orifices, noses and gums.
Death can follow quickly, though rapid medical care can greatly increase the chances of survival. Since it was discovered, the virus has killed an average of half of those it infects, with previous outbreaks killing between 24 per cent and 88 per cent of patients.
Marburg virus was first identified in 1967 after 31 people were infected and seven died in simultaneous outbreaks in Marburg and Frankfurt in Germany and Belgrade in Serbia.
The outbreak was traced to African green monkeys imported from Uganda.
Tanzania, in East Africa, last week announced its own outbreak of the Marburg virus. Health officials said it was too early to tell if the outbreaks, about 3,220km apart, are linked.