The aim of this study was to evaluate the clinical significance of the association of Streptococcus equi subsp zooepidemicus (SEZ) and Mycoplasma spp. bacteria in equine respiratory samples. In contrast to β-hemolytic streptococci and especially SEZ that are well-recognized pathogens of horses lower respiratory tract, Mycoplasma spp. are still regarded as potential opportunistic pathogens with no clear demonstration so far. Our study was conducted using 715 tracheal washes of horses with suspicion of respiratory disease sampled between 2020 and 2021. Classical bacteria were isolated and quantified by cell count, and identified using MALDI-TOF, while mycoplasmas were detected by both culture and real-time PCR, after an enrichment procedure, to consider their fastidious growth in vitro. None of the tested samples was considered polycontaminated (define as more than three bacterial species detected, in addition to Mycoplasma spp). The statistical significance of Mycoplasma spp. and other bacteria differences in prevalence were calculated using Chi-square test, enumeration by Mann-Whitney and Odds ratios by logistic regression. We noted that 123 samples were positive for SEZ and 114 for Mycoplasma spp., corresponding to a prevalence of 17% and 16%, respectively. Moreover 52 samples were positive for both bacteria. Prevalence of SEZ was significantly greater for Mycoplasma spp. positive samples (46% vs 12%; P = <0.0001). For SEZ-positive samples, no significant difference was observed in bacterial load whatever the presence of Mycoplasma spp. (P=0.23). The main clinical signs of respiratory diseases, namely dyspnoea, coughing, nasal discharge and hyperthermia, were recorded for 435/715 (60%) horses by the veterinarians during clinical examinations. Logistic regression was performed to each symptom in order to compare the population infected by SEZ and Mycoplasma spp. (SEZ+ / M+) and the population infected by SEZ only (SEZ+ / M-) to negative SEZ horses (SEZ-). Nonsignificant results were found for dyspnoea and cough. Compared to the SEZ-population, SEZ+/ M+ (OR = 2.7; P=0.01) and SEZ+/ M-(OR = 3.8; P=0.001) populations were more likely to have nasal discharge. Only horses with a double tracheal infection of SEZ and Mycoplasma spp. (SEZ+ / M+) have a significant higher risk (OR = 3.6; P=0.004) of hyperthermia compare to SEZ negative population (SEZ-). Nevertheless, this elevated risk was not the effect of an increased amount of SEZ in Mycoplasma positive horses. Our data suggest that Mycoplasma spp. appear to be a potentiator of infection when found in conjunction with SEZ in horses. The monitoring of these two species should be recommended when respiratory bacterial infection is suspected and particularly when nasal discharge and hyperthermia is present.