AbstractVegetation optical depth (VOD) satellite microwave retrievals provide significant insights into vegetation water content and responses to hydroclimatic changes. While VOD variations are commonly linked to dry biomass and live fuel moisture content (LFMC), the impact of canopy temperature (Tc) remains overlooked in large‐scale studies. Here, we investigated the impact of Tc on L‐band (1.4 GHz) and X‐band (10.7 GHz) VOD at diurnal and seasonal timescales. Synthetic benchmark VOD was created using realistic fields of Tc, LFMC, and biomass in an electromagnetic model. Perturbation experiments revealed that Tc strongly affects diurnal VOD variations at both L‐band and X‐band. Seasonally, while biomass emerges as the largest contributor to VOD variations in 70% (at X‐band) and 90% (at L‐band) of our study region, Tc and LFMC still play substantial roles. The findings stress the importance of refining retrieval algorithms to distinguish Tc, LFMC, and biomass effects for future VOD applications in ecohydrology.