Rapid detection of microbial contamination in conventional and alternative fuels is hampered by the lack of sensitive and cost-effective assays to detect total and specific microorganisms in fuels. Here, we report a simple and highly sensitive TaqMan quantitative polymerase chain reaction (qPCR) assay for universal detection and quantification of fungi, bacteria, and archaea in fuel in a single multiplexed reaction. Universal primers and probes targeting conserved regions of the 16S and 18S rRNA genes were designed and validated for specific amplification of total fungi, bacteria, and archaea in fuel. The assay is able to detect as low as 10 pg of fungal and bacterial DNA. The combination of a simple liquid-liquid extraction to recover cells from fuel with a freeze-and-heat method to release DNA for direct qPCR amplification eliminates the need for DNA extractions from contaminated samples, thus making the assay much faster, inexpensive, and less laborious. The universal microbial multiplex qPCR assay demonstrated a high capacity to detect and quantify a wide range of microbial contaminants. The assay was validated to accurately detect and quantify the intended microorganisms in the presence of high levels of nontarget DNA and in fuel from field samples. This robust microbial qPCR assay can be applied to microbial detection in environmental and industrial settings to facilitate risk assessment and mitigation of microbial contamination.