<p>PCR-based detection of pathogenic viruses and indicators in water is often hindered by naturally occurring substances that inhibit reverse transcription (RT) and PCR. Selecting RT-PCR assays tolerant to these inhibitors is essential. The degree of RT-PCR inhibition depends on the reaction mix composition and the specific inhibitors present. This study investigated the impact of RT-PCR inhibitors by targeting five viruses in 20 surface water samples containing the inhibitors under various conditions, including changes in sample volumes, RT-PCR reagents, primers and TaqMan probes, and the presence or absence of an inhibition-reduction method (SD treatment). The results revealed significant variability in detection frequencies and concentrations based on reagents and conditions. For example, one virus was detected in only one sample using one assay but in 16 samples using another. Differences exceeding 1 log₁₀ in detected concentrations were observed between assays. One RT-PCR reagent demonstrated higher detection frequencies and concentrations than others. However, its superiority was not consistent and depended on the targeted virus or primers and probes. Increasing the RNA extract volume amplified inhibitory effects, which also differed across reagents and viruses. These findings underscore the importance of selecting reagents, including primers and probes, that are compatible with the inhibitors present in samples. Optimizing test volumes is also critical for reliable virus detection. Furthermore, selecting an appropriate process control and its detection method based on their sensitivity to inhibitory effects is vital to ensuring accurate results.</p>