That included US concern for the imprisonment of Mam Sonando, owner of Beehive Radio, one of the few independent broadcasters in the country.
Cambodian officials have said numerous reports of human rights abuses ahead of Obama’s visit were meant to discredit the government.
A government spokesman dismissed the letter, saying it did not reflect the true situation in Cambodia.
This has led to increased reports of rights abuses in host countries, particularly Malaysia, where young Cambodian are sent to work as maids.
The two women were arrested in early September and accused of inciting violence and criminal activity among demonstrators.
The European Union on Friday called on Cambodia to immediately release jailed Beehive Radio owner Mam Sonando, while condemning political court actions and ongoing rightrs abuses.
Canadian journalist Olesia Plokhii was one of two journalists from the Cambodia Daily who went with environmentalist Chut Wutty to the remote jungle mountains of Koh Kong province in April.
Koh Kong provincial court has handed a commuted sentence of six months to the man alleged to have shot the military policeman who killed environmentalist Chut Wutty in April.
US Embassy spokesman Sean McIntosh told VOA Khmer in an interview Friday the US would continue to monitor the situation.
Mam Sonando has been in jail since his arrest in July and was handed a 20-year prison sentence earlier this month.
The gathering underscored the myriad fronts of unrest the government is currently facing, due to land grabs, forced evictions and other rights abuses.
Local analysts say such continued reactions from Hun Sen, who often blasts the UN from his pulpit, are bad for the image of the country.