In transit agencies, both route agents and administrative workers are exposed to psychosocial factors, which may affect their mental health. The present cross-sectional study was conducted with a sample of 131 transit agency workers in Esmeraldas (Ecuador) to determine the prevalence of mental distress associated with working conditions. The Goldberg Mental Health Questionnaire (GHQ12) with a cut-off point > 3 (dichotomous scoring approach) was considered. Moreover, EPI INFO software (V7) was used for data analysis to obtain chi-square, Fisher, crude and adjusted logistic regression tests. One of the study’s main findings was that the probability of suffering mental distress is not statistically associated with the type of work performed. Hence, the prevalence of mental distress was found to be 51.47% for route agents and 50.88% for administrative staff. On the other hand, the results of the logistic model show that the sex of the worker does have a significant effect as a predictor variable for mental distress. Thus, being female was a risk factor that increased the probability of suffering stress by 186% (adjusted OR: 2.86; 95% CI: 1.17–6.98) compared to male workers in the transit agency analyzed (confidence level of 95%).