In Nigeria and many other developing countries of the world, the incidence of mycotoxin- contamination of foods and food products has attracted attention and stirred a lot of concern for food safety. This work aims at detection of aflatoxigenic and ochratoxigenic synthetic genes from fungal isolates of palm oil as a veritable means for the evaluation of foods for possible mycotoxin contamination.  In this study, fungal isolates from palm oil samples collected from the five states of South-east geopolitical zone in Nigeria were screened for  aflatoxin and ochratoxin biosynthetic genes using Multiplex Polymerase Chain Reaction (mPCR). The assay relied on three sets of primers that amplify aflatoxgenic Aspergillus, ochratoxigenic Aspergillus and Penicillium species under optimized PCR conditions. Optimum multiplex PCR assay was standardized for simultaneous detection of toxigenic Aspergillus and ochratoxin producing Penicillium species targeting AflR, AflS and pks genes involved in aflatoxin and ochratoxin metabolic pathways respectively. AflR primer pair gave specific amplification for aflatoxigenic A. flavus but did not give amplification for A. niger and P. chrysogenum. While AflS and pks gave amplification for only aflatoxigenic and ochratoxigenic A. niger and P. chrysogenum. In the evaluation and monitoring of mycotoxin-producing fungi during the processing of food and feed commodities, Multiplex PCR approach could be a veritable tool to supplement the conventional analytical techniques.