Supplementary data for the paper "Wang S, Luo H. Dating the bacterial tree of life based on ancient symbiosis. Systematic Biology. 2024". Supplementary figures (Figs. S1-S19), tables (Tables S1-4), notes (Notes S1-S5), and datasets (Data S1-S5). Abstract Obtaining a timescale for bacterial evolution is crucial to understanding early life evolution but is difficult owing to the scarcity of bacterial fossils and the absence of maximum age constraints of the available fossils. Here, we introduce multiple new time constraints to calibrate bacterial evolution based on ancient symbiosis. This idea is implemented using a bacterial tree constructed with mitochondria-originated genes where the mitochondrial lineage representing eukaryotes is embedded within Proteobacteria, such that the date constraints of eukaryotes established by their abundant fossils are propagated to ancient co-evolving bacterial symbionts and across the bacterial tree of life. Importantly, we formulate a new probabilistic framework that considers uncertainty in inference of the ancestral lifestyle of modern symbionts to apply 19 relative time constraints (RTC) each informed by host-symbiont association to constrain bacterial symbionts no older than their eukaryotic host. Moreover, we develop an approach to incorporating substitution mixture models that better accommodate substitutional saturation and compositional heterogeneity for dating deep phylogenies. Our analysis estimates that the last bacterial common ancestor (LBCA) occurred approximately 4.0-3.5 billion years ago (Ga), followed by rapid divergence of major bacterial clades. It is generally robust to alternative root ages, root positions, tree topologies, fossil ages, ancestral lifestyle reconstruction, and gene sets, among other factors. The obtained time tree serves as a foundation for testing hypotheses regarding bacterial diversification and its correlation with geobiological events across different timescales.