Assessing canine semen quality helps to detect infertility in males, but identifying factors that influence canine semen quality is a complicated task. The objective of this study was the assessment of the potential influence of bacteria found in canine semen samples on the characteristics of dogs’ semen. In this study, semen samples were collected manually from 30 dogs and subjected to a comprehensive examination. The results of sperm motility, concentration, viability, and morphology were statistically analysed in relation to the number of bacteria in the semen (CFUs/mL) and the seminal microbiota. Samples with an increased bacterial count per millilitre were associated with lower-quality sperm motility (p < 0.05). The most frequently isolated bacterial genera from the analysed semen samples were Staphylococcus spp. (26.0%), Corynebacterium spp. (17.8%), and Streptococcus spp. (16.4%). The presence of β-haemolytic Escherichia coli bacteria was linked to suboptimal semen samples, characterised by significantly reduced semen viability and a lower proportion of morphologically normal spermatozoa (p < 0.05). Corynebacterium spp. was associated with reduced bacterial load and superior semen quality (p < 0.01). These findings highlight the importance of bacterial cell counts and microbiota diversity in relation to various factors influencing canine semen quality, providing a more comprehensive understanding of canine reproductive well-being.