Multiplex polymerase chain reaction (mPCR) assay is a nucleic acid amplification method that is considered reliable and practical means for several pathogen detections in a single reaction, especially when multiple pathogens are suspected. In this study, a novel mPCR assay was validated for the detection of four notifiable diseases in cattle, including foot and mouth disease (FMD), Bovine viral diarrhea (BVD), Bluetongue (BT), and Hemorrhagic Septicemia (HS). The assay was operated in a two-step procedure. The first one was a reverse transcription of viral RNA, then mPCR of viral cDNA and bacterial DNA. The optimized mPCR was applied on blood (26) and vesicular epithelium (10) samples collected from 26 clinically infected animals from three governorates (Qalubia, Sharkia, and Gharbia). mPCR detected at least 10 pg of microbial nucleic acid extracted from the local isolates. The mPCR results showed that 22/26 (84.6%) of clinically infected animals were positively infected by single or dual infection. Mixed infection of FMDV and Pasteurella multocida was recorded in 11 animals (42.3%), while single FMDV infection was recorded in 5 animals (19.2 %). Single BVDV infection was detected in 5 animals (19. 2 %) and dual infection with FMDV in 1 animal (3.8%). Notably, BTV was not detected in any of the clinical samples. The assessed mPCR was a rapid, accurate, and sensitive test for diagnosing single and mixed infections in cattle and could be used to screen the notifiable diseases affecting cattle.