Infectious diseases (e.g., coronavirus disease 2019) dramatically impact human life, economy and social development. Exploring the low-cost and energy-saving approaches is essential in removing infectious virus particles from indoors, such as in classrooms. The application of air purification devices, such as negative ion generators (ionizers), gains popularity because of the favorable removal capacity for particles and the low operation cost. However, small and portable ionizers have potential disadvantages in the removal efficiency owing to the limited horizontal diffusion of negative ions. This study aims to investigate the layout strategy (number and location) of ionizers based on the energy-efficient natural ventilation in the classroom to improve removal efficiency (negative ions to particles) and decrease infection risk. Three infected students were considered in the classroom. The simulations of negative ion and particle concentrations were performed and validated by the experiment. Results showed that as the number of ionizers was 4 and 5, the removal performance was largely improved by combining ionizer with natural ventilation. Compared with the scenario without an ionizer, the scenario with 5 ionizers largely increased the average removal efficiency from around 20% to 85% and decreased the average infection risk by 23%. The setup with 5 ionizers placed upstream of the classroom was determined as the optimal layout strategy, particularly when the location and number of the infected students were unknown. This work can provide a guideline for applying ionizers to public buildings when natural ventilation is used.