Introduction Pre-hospital care personnel, including paramedics and emergency medical technicians (EMTs), are crucial frontline responders in emergency medical situations. They are critical in delivering timely medical assistance to individuals in emergencies. However, the demanding nature of their work can subject them to significant job stress, which may adversely affect their well-being and job performance. Objectives This study aimed to assess the levels of job stress and explore associated factors among pre-hospital care providers in Addis Ababa. Methodology A cross-sectional study was conducted at a healthcare facility, involving 134 ambulance healthcare providers. Participants were selected through a simple random sampling technique. Data collection employed pretested, self-administered questionnaires utilizing a nursing stress scale. Descriptive statistics were utilized to summarize the findings. Logistic regression analysis was employed to ascertain associations between variables while controlling for potential confounders. The strength of the association between dependent and independent variables was evaluated using odds ratios with a 95% confidence interval. Result Of the 134 study participants, 47% (95% CI: 38.2, 55.8) of them had job stress. Notably, several factors were significantly associated with job stress among pre-hospital healthcare providers working in ambulances. Conflict with leaders (AOR = 5.07; 95% CI: 2.98, 12.53), fear of mistakes (AOR = 8.22; 95% CI: 1.86, 36.34), lack of resources (AOR = 11.06; 95% CI: 5.19, 22.41), overloaded with ambulance care (AOR = 18.94; 95% CI: 4.33, 38.73) and inadequate information from dispatchers (AOR = 7.35; 95% CI: 3.32, 16.62) were identified as significant associated factors of job stress among these providers. These findings highlight the critical need to address leadership conflicts, fear of errors, and communication deficiencies to mitigate job stress among pre-hospital healthcare providers working in ambulances. Conclusion This study highlights the prevalence of job stress among pre-hospital healthcare providers working in ambulances, and found it as a significant problem. Notably, conflict with leaders, fear of mistakes, lack of resources, work overload and inadequate information from dispatchers emerged as significant associated factors contributing to job stress among pre-hospital health care providers. Efforts to foster better communication channels between providers and dispatchers, promote supportive leadership practices, and implement error-reduction strategies are essential. By addressing these challenges, healthcare organizations can create a more conducive work environment that supports the mental and emotional health of pre-hospital healthcare providers.