In contrast to capital control strategies (i.e., investments in new technology), the potential of operational control strategies (e.g., vehicle scheduling optimization) to reduce the health and climate impacts of the emissions from public transportation bus fleets has not been widely considered. This case study demonstrates that heterogeneity in the emission levels of different bus technologies and the exposure potential of bus routes can be exploited though optimization (e.g., how vehicles are assigned to routes) to minimize these impacts as well as operating costs. The magnitude of the benefits of the optimization depend on the specific transit system and region. Health impacts were found to be particularly sensitive to different vehicle assignments and ranged from worst to best case assignment by more than a factor of 2, suggesting there is significant potential to reduce health impacts. Trade-offs between climate, health, and cost objectives were also found. Transit agencies that do not consider these objectives in an integrated framework and, for example, optimize for costs and/or climate impacts alone, risk inadvertently increasing health impacts by as much as 49%. Cost-benefit analysis was used to evaluate trade-offs between objectives, but large uncertainties make identifying an optimal solution challenging.