"This is a second round of a dirty conspiracy," the 60-year-old told reporters.
Anwar on Monday filed a defamation lawsuit against the aide.
Tuesday's complaint was filed against Musa Hassan, the inspector general of police, and Abdul Gani Patail, the attorney general, at a police station in Shah Alam, the capital of Selangor state that adjoins Kuala Lumpur.
Anwar was fired as deputy prime minister in 1998 after being accused of sodomising his driver and an aide.
He was tried and convicted but a higher court overturned the conviction in 2004. Still, a related abuse of power conviction kept him banned from holding political office until April, a month after the general elections were held.
During that election, Anwar, a former deputy premier, made a dramatic return to Malaysian politics.
His opposition alliance made a surprisingly strong showing in the polls, grabbing one-third of the seats in parliament and undermining the coalition's long-time hold on power.
By law, Anwar has to be a lawmaker before he can take the prime minister's job.
Anwar said he had originally planned to announce on Tuesday details of where he will contest a by-election.
"This has been derailed for a few days" because of the sodomy accusation, he said.
Saiful Izham Ramli, a member of the policy board of Anwar's Keadilan party, told the AFP news agency that the group will hold a campaign and rally on Tuesday night.
"We are now going on an offensive," he said. "Now we have a game plan."