Asim Hussain and other accused were produced in an accountability court where Judge Saad Qureshi read out the charges against them. They all pleaded not guilty.
The court summoned the prosecution witnesses with direction to record their testimonies in the next hearing of the case on May 14.
Few months after Hussain was arrested by the paramilitary rangers, former President Zardari went abroad and has remained there since.
Hussain has also been accused of receiving commissions from a fertiliser cartel for an "exploitative price hike".
Another charge related to "black marketing and fraud with the public" in the name of a charity hospital.
The NAB said that Hussain while he was federal petroleum minister had deprived the state of Rupees 462.5bn from 2010 to 2013 - Rupees 450 billion through the fertiliser scam, Rupees 9.5 billion through land fraud and Rupees 3 billion through money laundering.