Various microorganisms, including Mycoplasma spp., have been reported in canine ejaculate. The impact of these microorganisms on semen quality remains unclear. This study included 63 male intact healthy dogs aged 1–8 years. One dog exhibited azoospermia, indicating a relatively low incidence of this condition. Interestingly, 36.5% of the examined dogs tested negative for both aerobic bacteria and mycoplasmas, while 12.7% tested positive for bacterial presence. Additionally, 60.3% of the dogs tested positive for Mycoplasma spp. using PCR, with most carrying 1–2 Mycoplasma species. We found no significant difference in semen characteristics between Mycoplasma-positive and -negative dogs. The detection of Mycoplasma was not significantly linked to the presence of bacteria in semen. All the microorganisms identified were classified as saprophytic flora. Our findings indicate that Mycoplasma spp. is common in canine ejaculate. Semen quality parameters were not correlated with the presence of Mycoplasma spp. in semen. Mycoplasma HRC689 was the most common species. Some dogs exhibited no presence of aerobic bacteria or mycoplasmas in their semen. Our study highlights the common presence of Mycoplasma spp. in canine ejaculate. Semen quality shows no correlation with Mycoplasma presence. Some canine ejaculate is sterile. Our findings suggest the existence of undescribed species of canine mycoplasmas, necessitating advanced diagnostic techniques like NGS for their identification.