Species trees, which depict the evolutionary relationships among organisms, underlie many evolutionary studies. Phylogenomics, the use of genome-scale datasets for phylogenetic inference, is the current gold standard for species tree inference. The development, maintenance, and execution of phylogenomic workflows is challenging, requiring programming, data management skills, and familiarity with changing best practices. We introduce Orthoflow, a software wherein a single command automatically conducts end-to-end phylogenomic analysis—orthology inference and identification of phylogenomic markers, quality control, data matrix construction, diagnostics, and tree inference using supermatrix and supertree methods from multiple input data formats. To demonstrate the utility of Orthoflow, we successfully recapitulate the evolutionary relationships among 24 yeast species. Orthoflow increases the accessibility of researchers to conduct rigorous phylogenomic analysis flexibly. Orthoflow is freely available from PyPI (https://pypi.org/project/orthoflow/), Bioconda (https://anaconda.org/bioconda/orthoflow) and GitHub (https://github.com/rbturnbull/orthoflow) under the Apache License 2.0.