Gangliosides are essential glycosphingolipids critical in neurodevelopment and cell signaling. Traditionally sourced from animal tissues, their production raises ethical concerns and faces challenges in scalability and cost. Chemoenzymatic methods have emerged as alternatives but lack flexibility and broad industrial applicability of microbial systems. However, complete microbial biosynthesis remains challenging due to the complexity of reconstructing the biosynthetic pathway in non-native hosts. We report the first successful complete microbial synthesis of gangliosides by engineering the industrial filamentous fungus Ashbya gossypii. Using modular metabolic engineering, we heterologously expressed human and yeast enzymes to reconstruct a functional ganglioside biosynthetic pathway. Pathways for producing activated N-acetylneuraminic acid, lactosylceramide, and sialylated intermediates were integrated, yielding GM3 and GD3 at milligram-per-liter levels. These titers were further enhanced by introducing a heterologous Leloir pathway for galactose metabolism. This work represents a foundational advance in microbial glycoengineering, offering a scalable, animal-free microbial platform for ganglioside production with broad applications.