The relationship between modelled root zone soil moisture (SM) and black truffle production in the Iberian Peninsula was studied. Previous works have investigated the influence that precipitation exerts on truffle yield highlighting the importance of water for the growth of black truffle. However, SM had not been used until now due to the lack of suitable databases. The SM series from the LISFLOOD hydrological rainfall–runoff model was used in this study. Annual black truffle yield series from 175 locations in Spain was correlated with SM for the period 1991–2012. For this, different approaches were applied considering daily, weekly and monthly temporal scales. The same analysis was carried out using precipitation data to compare the behaviors of both variables related to truffle production variability. The results obtained show critical periods in terms of soil water content in summer (June–September) and during October–November months. Moreover, a clear delay between precipitation and SM influence on black truffle was observed. The results obtained in this study highlight the importance of SM for black truffle production, since this variable truly expresses the available water for this fungus, which completes its entire life cycle living below ground.