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The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between the two strains in cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems.