Ambrosia beetles rely on vertically transmitted fungal cultivars for nutrition, yet empirical studies reveal a persistent paradox: early galleries often host multiple fungal associates, whereas mature systems exhibit strict one-beetle–one-fungus specificity. The mechanisms driving this transition remain unresolved. Here we develop an integrative eco-evolutionary framework combining deterministic competition (Lotka–Volterra dynamics), stochastic drift, and severe mycangial transmission bottlenecks to quantify the stability and turnover of fungal symbionts. Across >50,000 simulation replicates, we show that multi-fungal coexistence is inherently transient under biologically realistic conditions. Even minimal competitive asymmetries (<5%) yield deterministic exclusion; repeated bottlenecks amplify drift and accelerate diversity loss; and weak but persistent selection ensures long-term fixation of the superior symbiont over hundreds of generations. Replacement by novel fungi follows a strict invasion threshold (s > 1/(2N)), consistent with classical fixation theory and matching empirical patterns of historical symbiont turnover. Multispecies communities collapse even more rapidly, persisting only over short ecological timescales. Together, these results provide a mechanistic explanation for the pervasive one-fungus specificity observed across ambrosia beetles. By unifying ecological competition, demographic stochasticity, and evolutionary stability theory, this work offers a general framework for understanding partner fidelity, symbiont filtering, and lineage turnover in insect–microbe mutualisms.