This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity’s impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.