Due to the emerging threat conditions in the work environment, firefighters are at a high risk of exposure to not only toxic substances but also biological agents in the dayroom and during emergency runs. The aim of this study is to evaluate firefighter (career and volunteer) knowledge and practice behaviors on infection control. This study surveyed 444 firefighters (210 career, 234 volunteer) in rural Northwestern Kentucky. The self-reported survey focused on individual characteristics, knowledge on exposure incident control, precautionary actions, and personal protections. We evaluated the descriptive characteristics of knowledge and practice scores stratified by firefighter groups (career and volunteers). The associations between infection control training received (yes/no) and firefighter knowledge and practice scores were also examined. Firefighters who were trained on infection control prevention had significantly higher knowledge scores (M = 63.7, SD = 13.4 vs. M = 59.7, SD = 15.9; p = 0.012). Volunteer firefighters exhibited better infection control practice behaviors than career firefighters (M = 70.6, SD = 13.0 vs. M = 67.4, SD = 11.1; p = 0.05). Firefighters who followed infection control guidelines (M = 69.5, SD = 11.9 vs. M = 58.1, SD = 9.9; p = 0.012) and expressed need for a comprehensive training on personal protective equipment (PPE) selection (β = 3.41, SE = 1.54, aOR = 30.22, 95% CI: 1.47–620.87; p = 0.028) had significantly higher practice scores compared to those who did not. The study results have policy implications for infection prevention and control (IPC) in rural fire departments, both career and volunteer. A review of infection control policies is needed, especially as it relates to training and practice behaviors during emergency calls and in the dayroom. Results also suggest the need to develop strategies to improve the culture of PPE use and training on the selection of PPEs appropriate to the emergency response type.