Fresh air ventilation has been identified as a widely accepted engineering control effective at diluting air contaminants in enclosed environments. The goal of this study was to evaluate the effects of selected ventilation measures on air change rates in school buses. Air changes per hour (ACH) of outside air were measured using a well-established carbon dioxide (CO2) tracer gas decay method. Ventilation was assessed while stationary and while traversing standardized route during late autumn/winter months in Colorado. Seven CO2 sensors located at the driver’s seat and at passenger seats in the front, middle, and rear of the bus yielded similar and consistent measurements. Buses exhibited little air exchange in the absence of ventilation (ACH = 0.13 when stationary; ACH = 1.85 when mobile). Operating the windshield defroster to introduce fresh outside air increased ACH by approximately 0.5–1 ACH during mobile and stationary phases. During the mobile phase (average speed of 23 miles per hour (mph)), the combination of the defroster and two open ceiling hatches (with a powered fan on the rear hatch) yielded an ACH of approximately 9.3 ACH. A mobile phase ACH of 12.4 was achieved by the combination of the defroster, ceiling hatches, and six passenger windows open 2 inches in the middle area of the bus. A maximum mobile phase ACH of 22.1 was observed by using the defroster, open ceiling hatches, driver window open 4 inches, and every other passenger window open 2 inches. For reference, ACHs recommended in patient care settings where patients are being treated for airborne infectious diseases range from 6 to ≥12 ACHs. The results indicate that practical ventilation protocols on school buses can achieve air change rates thought to be capable of reducing airborne viral transmission to the bus driver and student passengers during the COVID-19 pandemic.