Cannabis plants are susceptible to microbial contamination, including fungi capable of producing harmful mycotoxins. The presence of these toxins in cannabis products poses serious health risks, particularly to immunocompromised people. This study evaluated the safety of dried cannabis buds intended for medicinal use by examining microbial contamination and residual mycotoxins through culture-based techniques, PCR/qPCR, and ELISA. Irradiation significantly reduced viable fungal and bacterial colony-forming units (CFUs) and eliminated culturable bacteria but did not achieve complete sterilization. Viable spores of toxigenic genera such as Aspergillus, Penicillium, and Fusarium persisted. Sanger sequencing of the PCR product of the sample's DNA generated using Internal Transcribed Spacer (ITS) primers identified dominant mycotoxigenic fungi such as Aspergillus, Cladosporium, Fusarium, and Penicillium in non-irradiated (NR) samples, while next-generation sequencing (NGS) revealed additional non-culturable species. PCR and qPCR studies detected biosynthetic genes for aflatoxins (Nor1), trichothecenes (Tri5), ochratoxins (PKS), and deoxynivalenol (DON) across all samples. While band intensity decreased post-irradiation, gene copy numbers remained comparable, suggesting DNA damage without full degradation. ELISA confirmed the presence of aflatoxin, ochratoxin, DON, and T2 toxins in both irradiated and licensed producer (LP) marketplace samples, with variable concentrations. LP samples showed lower microbial counts and mycotoxin gene abundance but still contained detectable residual DNA and toxins. These findings indicate that while irradiation lowers microbial loads, it does not eliminate mycotoxigenic fungi or their metabolites. Therefore, culture-based assays alone are insufficient for comprehensive safety assessments and must be complemented by molecular and immunological techniques. Given the persistence of toxigenic fungi and their toxins in irradiated and licensed products, we recommend stricter microbial safety standards, particularly for medicinal cannabis, which may put immunocompromised patients at higher risk. For high-risk pathogens like Aspergillus and Fusarium, which pose significant health concerns, a zero-tolerance threshold (<10 CFU/g) is advised, supported by stringent decontamination and testing.