<p>This study aimed to evaluate the level of strain experienced by professionals across 104 French assisted reproductive technology (ART) centers. A cross-sectional survey was conducted from November 2023 to January 2024, using an online questionnaire based on Karasek’s and Siegrist’s models to assess psychological demands, decision latitude, social support, and the balance between efforts and rewards. Additional questions collected personal and professional details. Job strain was defined by high demands and low decision latitude, while iso-strain also included low social support. Effort-reward imbalance (ERI) referred to a situation where individuals experience high effort in their work but perceive the rewards as insufficient. The study included 464 participants (13% participation rate), comprising biologists (<i>n</i> = 124, 26.7%), gynecologists (<i>n</i> = 129, 27.8%), technicians (<i>n</i> = 107, 23.1%), secretaries (<i>n</i> = 51, 11.0%), midwives (<i>n</i> = 31, 6.7%), and other professions (<i>n</i> = 22, 4.7%). We found that 35.7% of professionals in French ART centers experienced job strain, with 20.9% in an iso-strain situation and 14.9% showing an imbalance between efforts and rewards. Multivariate logistic regression analysis revealed a significant impact of occupational roles on job strain, with secretaries (OR 9.76 [3.70; 25.78]) and technicians (OR 8.62 [3.45; 21.53]) facing a higher risk compared to gynecologists. A strong correlation was found between perceived work stress or job satisfaction and the risk of job strain, iso-strain, and ERI. In conclusion, our study revealed that over a third of French ART professionals experienced job strain, a condition linked to increased risks of stress, burnout, and health concerns. Secretaries and technicians appeared particularly vulnerable, highlighting the need for targeted interventions. Future research should aim to identify specific stressors and protective factors to help create tailored support programs that promote staff well-being.</p>