Michael Danby, chairman of parliament's foreign affairs subcommittee, delivered the letter to the Malaysian high commissioner, Salman Bin Ahmad, yesterday.
The letter says: "It should be made known to the Malaysian government that in our opinion global esteem for Malaysia will be affected by these charges against Mr Anwar."
The letter further states: "Many friendly observers of Malaysia find it difficult to believe that a leading opposition voice could be charged with sodomy a second time, and so soon after his party made major gains in national elections."
Most of the 50 signatories are Labor members, including two frontbenchers, parliamentary secretaries Gary Gray and Laurie Ferguson. The Deputy Speaker of the House of Representatives, Anna Burke, also signed the letter.
On the Liberal side, environment spokesman Greg Hunt is the most senior signatory, along with former opposition leader Malcolm Turnbull.
Mr Anwar was first charged with sodomy in 1998. He was convicted, but in 2004 Malaysia's Federal Court overturned his conviction.
Mr Anwar was a former deputy prime minister and finance minister under Malaysia's long-serving former leader, Mahathir Mohammed. The two fell out bitterly in the wake of the 1998 Asian financial crisis. Since he was released from jail, Mr Anwar has become the leader of the Malaysian opposition, which made sweeping gains in Malaysia's last federal and state elections.
Such a strong pro-Anwar intervention by so many, and such senior, Australian politicians could well once more inflame the Australia-Malaysia relationship, which went through turbulent times when Dr Mahathir and Paul Keating were their countries's respective leaders. Since then Australia has trodden softly to ensure Malaysian co-operation on trade, military and intelligence, counter-terrorism and regional efforts to stop people-smuggling.