A decline of various grapevines (Vitis vinifera L.) in the province of Doukkala in Morocco was observed in 2021. The causal pathogen was identified as Lasiodiplodia viticola based on morphological characteristics and phylogenetic analysis of the internal transcribed region (ITS), the β-tubulin gene (TUB) and calmodulin (cmdA). Koch’s postulates were confirmed by successful re-isolation of L. viticola from plants inoculated with the pathogen under controlled conditions. The disease was shown to be prevalent in Bni Hilal (71.43%), Laamria (60%), and Boulaouane (40%) districts, but was quasi-absent in Lmechrek. To understand the dominance of L. viticola as one of the grapevine trunk pathogens, effects of temperature (10–40 °C) and pH (pH 3–pH 12) on growth and sporulation were investigated. The species were able to grow in a range of temperatures ranging from 15 to 40°C and showed a higher growth rate at 35 °C. The fungus were also characterized by a broad optimum pH ranging between 3–12. This study is the first report dealing with L. viticola associated with grapevine trunk diseases in Morocco. Additional studies are therefore required to understand the high occurrence of this disease in vineyards, which is likely due to climate changes. A good understanding of this complex disease might help to develop a reliable and sustainable preventive control strategy.