In 2024, Israel reported its largest West Nile Virus (WNV) outbreak to date, with 934 confirmed cases and 73 deaths. Current diagnostic practice predominantly relies on serological detection of IgM antibodies as the first-line approach, with molecular testing considered complementary; however, cross-reactivity with co-circulating flaviviruses and prolonged IgM persistence complicate accurate case confirmation. Leveraging the extensive sample collection during this outbreak, we evaluated and compared the diagnostic performance of molecular and serological assays across multiple sample types. During the outbreak, the Israeli National Laboratory for Zoonotic Viruses received more than 2,000 samples from 919 WNV-positive cases, including whole blood (WB), serum, urine, and cerebrospinal fluid (CSF), examined by qRT-PCR and ELISA. WNV RNA detection rates were highest in WB (91%), followed by serum (82%), urine (71%), and CSF (53%), with WB remaining positive up to 52 days post-symptom onset, substantially exceeding the detection window of all other sample types. Of note, while WB RNA detection rates were similar between hospitalized patients and health maintenance organization (HMO) patients (91% vs. 92%), IgM seropositivity in serum was significantly lower in HMO-derived samples (69%) compared to hospital samples (91%), likely reflecting earlier disease stage at the time of community testing. These data suggest that for a substantial proportion of community cases, serological diagnosis alone is insufficient and molecular testing on WB is the more reliable approach, with important implications for timely outbreak surveillance. In a complementary and independent validation study on a well-characterized cohort of confirmed acute and past WNV cases, IgG avidity testing at an optimized cut-off of 35% achieved a negative predictive value of 96%, offering a rapid single-sample alternative to the labor-intensive neutralization assay currently required for diagnostic confirmation. Collectively, these findings support reassessing current WNV diagnostic algorithms, prioritizing WB-based molecular testing as the primary modality, complemented by IgG avidity as a serological tool in cases where molecular testing is no longer informative due to late presentation after symptom onset, together improving diagnostic accuracy and public health outbreak response.