There is an increasing role for bioinformatic and phylogenetic analysis in tropical medicine research. However, scientists working in low- and middle-income regions may lack access to training opportunities in these methods. To help address this gap, a 5-day intensive bioinformatics workshop was offered in Lima, Peru. The syllabus is presented here for others who want to develop similar programs. To assess knowledge gained, a 20-point knowledge questionnaire was administered to participants (21 participants) before and after the workshop, covering topics on sequence quality control, alignment/formatting, database retrieval, models of evolution, sequence statistics, tree building, and results interpretation. Evolution/tree-building methods represented the lowest scoring domain at baseline and after the workshop. There was a considerable median gain in total knowledge scores (increase of 30%, p< 0.001) with gains as high as 55%. A 5-day workshop model was effective in improving the pathogen-applied bioinformatics knowledge of scientists working in a middle-income country setting.